Methods for delivering compounds into a cell

ABSTRACT

The present invention is directed, inter alia, to a method for delivering a compound into a cell comprising administering to the cell the compound to be delivered, an organic halide, and/or a carrier. Ultrasound may also be applied, if desired.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/785,661, filed Jan. 17, 1997, which in turn is a continuation-in-partof U.S. application Ser. No. 08/640,554, filed May 1, 1996, thedisclosures of each of which are hereby incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

This invention relates to the field of intracellular delivery, inparticular, to the use of organic halides and/or ultrasound tofacilitate the delivery of a compound into a cell.

BACKGROUND OF THE INVENTION

Cells are the basic structural and functional units of all livingorganisms. All cells contain cytoplasm surrounded by a plasma, or cell,membrane. Most bacterial and plant cells are enclosed in an outer rigidor semi-rigid cell wall. The cells contain DNA which may be arrangedin 1) a nuclear membrane or 2) free in cells lacking a nucleus. Whilethe cell membrane is known to contain naturally occurring ion channels,compounds that are therapeutically advantageous to cells are usually toolarge to pass through the naturally occurring ion channels. Conventionalinterventional methods of delivery of compounds into cells have proveddifficult in view of the need for the compounds to pass through the cellmembrane, cell wall, and nuclear membrane.

Molecular biology has resulted in mapping the genomes of many plants andanimals including the mapping of much of the human genome. The potentialfor advances in the understanding of the genetic basis of diseases isgreat, as is the potential for the development of therapies to treatsuch diseases. However, to fully take advantage of these advancementsand treatment therapies, methods are needed which will allow for thedelivery of desired compounds into the target cells. Accordingly,researchers have undertaken the development of a variety ofintracellular delivery methods for inserting genes and other compoundsinto both plant and animal cells.

For example, calcium phosphate DNA precipitation has been used todeliver genetic material into cells in cell culture. However, onedrawback of this method is that the resultant efficiency of transfection(delivery of the genetic material into the cells) and subsequent geneexpression has been very low.

Improved transfection has been attained using viral vectors, e.g.,adenovirus and retrovirus, but again, difficulties with gene expressionhave persisted. In addition, substantial concerns regarding antigenicityand the potential of mutant viruses and other possible deleteriouseffects exist.

Liposomes, manufactured more easily than viral vectors, have shownpromise as gene delivery agents. Liposomes have less biological concerns(in that, for example, they are generally non-antigenic) but theefficiency of transfection and gene expression using liposomes hastypically been lower than with viruses.

Gene guns, wherein genes are attached to heavy metal particles such asgold, have been used to fire the particles at high speed into cells.However, while gene guns have resulted in gene expression in culturesystems, they have not worked well in vivo. Furthermore, the blast ofheavy metal particles may cause damage to the cells and may result inthe introduction of undesirable foreign materials, e.g. gold particlefragments, into the cells.

Electroporation is another method of delivering genes into cells. Inthis technique, pulses of electrical energy are applied to cells tocreate pores or openings to facilitate passage of DNA into the cells.However, electroporation may damage cells, and furthermore has not beenshown to be highly effective in vivo.

Various publications disclose the use of lithotripsy shock waves foreffecting intracellular gene transfer, as well as the delivery of othercompounds, including, for example, Delius, M., et al., “ExtracorporealShock Waves for Gene Therapy,” Lancet May 27, 1995, 345:1377; Lauer, U.,et al., “Towards A New Gene Transfer System: Shock Wave-Mediated DNATransfer,” J Cell Biochem 1994, 16A:226; Gambihier, S., et al.,“Permeabilization of the Plasma Membrane of L1210 Mouse Leukemia CellsUsing Lithotripter Shock Waves,” J Membr Biol 1994, 141:267-75; andMobley, T. B., et al., “Low Energy Lithotripsy with the Lithostar:Treatment Results with 19,962 Genal and Ureteral Calculi,” J Urol 1993,149:1419-24. Lithotripsy delivers energy in the range of 200-380 bars,and a frequency of 60-120 Hz, but may be as high as 1200 to 1300 bars.The energy and frequency ranges are typically painful to a patient andthus usually require patient sedation. Lithotripsy machines are largeand bulky and are typically cost prohibitive. Lauer et al. disclose thedelivery of 250 shock waves at 25 kV with a lithotripter to deliverplasmid DNA which expressed hepatitis B virus surface proteins in a HeLacell suspension.

Gambihler et al. (cited above) teach the permeabilization of mouse cellsin vitro to deliver dextrans. The lithotripter shock waves are deliveredat 25 kV, at a discharge rate of 60/min. Mobley et al. (cited above)disclose the use of lithotripsy to treat renal and ureteral stones. Theshock wave pressure was 200 to 380 bar and a generator range of 10 to 29kV.

Zhang, L., et al., “Ultrasonic direct gene transfer The Establishment ofHigh Efficiency Genetic Transformation System for Tobacco,” Sci Agric.Sin. 1991, 24:83-89 disclose increased gene expression by tobacco usingcontinuous wave ultrasound at 0.5 W/cm² for 30 minutes. Zhang et al. donot disclose the ultrasound frequency. The high energy level is in arange necessary for poration to result in the cell wall of tobaccoplants.

Rubin, et al., 31st Annual Meeting of the American Society of ClinicalOncology, May 20-23, 1995, disclose the injection of hepatic tumors witha plasmid/cationic lipid complex with ultrasound guidance. Ultrasound isdisclosed as a visual guide to monitor the injection of the tumors,rather than as an aid to deliver the complex to the liver tumors.

The present invention provides new and/or better methods for deliveringcompounds, including genetic material, into a cell. The methods of thepresent invention may provide a significant advantage over prior artmethodology, in that enhanced levels of intracellular delivery, and inthe case of nucleotides, gene expression, may be achieved. In addition,the process of the present invention may be performed in cell lineswhich may be otherwise resistant to intracellular delivery and geneexpression using other conventional means. These and/or other aspects ofthe present invention will become apparent from the further discussionsherein.

SUMMARY OF THE INVENTION

The present invention is directed, inter alia, to a method fordelivering a compound into a cell comprising administering to the cell acomposition which comprises the compound to be delivered and an organichalide.

In addition, the invention provides a method of treating a patientcomprising administering to a patient a composition comprising atherapeutically effective amount of a compound and an organic halide.

The subject invention provides a method of effecting the expression of anucleotide sequence in a cell comprising administering to said cell acomposition which comprises a nucleotide sequence and an organic halide.

If desired, the compositions may further comprise a carrier. Inaddition, the method of the invention may further comprise theapplication of ultrasound, as desired.

The present invention is also directed to a method for delivering acompound into a cell comprising administering to the cell the compoundto be delivered, or a composition comprising the compound to bedelivered, and applying ultrasound.

Further, the invention pertains to a method of treating a patientcomprising administering to a patient a therapeutically effective amountof a compound, or a composition comprising a therapeutically effectiveamount of a compound, and applying ultrasound.

Moreover, the subject invention provides a method of effecting theexpression of a nucleotide sequence in a cell comprising administeringto the cell a nucleotide sequence, or a composition which comprises anucleotide sequence, and applying ultrasound.

If desired, the composition may further comprise carrier.

Also included in the present invention are compositions and kitscomprising, for example, a therapeutically effective or diagnosticallyeffective amount of a compound to be delivered, an organic halide,and/or a carrier, and, in the case of a kit, optionally otherconventional kit components.

These, as well as other, aspects of the invention are set forth ingreater detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a test set-up for delivery of compounds to a cell invitro or ex vivo. In FIG. 1, A represents a standoff platform. B is asix well plate, with individual wells B′. C represents an ultrasoundgel, while D represents a therapeutic ultrasound transducer. Inaccordance with the invention, cells, the compound to be delivered, anorganic halide (if desired), and optionally a carrier are placed in thewells. Using ultrasound transducer D, ultrasound is then applied to cellculture plate B such that the standoff platform (A) is cut (G) undereach well (B′) for focusing ultrasound to the individual wells (B′).Ultrasound transducer D may also be employed for the in vivo delivery ofcompounds by applying transducer D, with ultrasound gel C, to a patientinstead of to a cell culture plate.

FIG. 2 displays the relationship between energy deposition, ultrasoundenergy intensity, and ultrasound duty cycle (pulse duration). The effectof attenuation as a function of tissue depth is also portrayed as wellas spatial peak temporal average power.

FIG. 3 is a map of the pCAT® control vector (GenBank accession numberX65321) (Promega, Madison, Wis.) used in the preparation of sequencesintroduced into cells in Examples 2 and 3.

FIG. 4 is a cutaway from FIG. 1, depicting one of the six wells (B′) ofa six well plate B. In FIG. 4, A is a portion of the standoff platform,B′ is one well of six well plate B, C is the ultrasound gel, D is theultrasound transducer, E represents the cells, and F representsmicrosphere carriers for the compound to be delivered.

FIG. 5 is a prototype second harmonic transducer that emits X and 2×frequencies and superimposes two beams at one focal point for enhancedultrasound effect. The transducer may be employed in conjunction with invitro, ex vivo and in vivo delivery of compounds and compositions inaccordance with the invention.

FIG. 6 is a map of the pCAT® basic vector (Promega, Montgomeryville,Pa.) used in the preparation of sequences introduced into cells inExample 10.

FIG. 7 depicts the results of the in vivo transfection studies in miceset forth in Example 23.

DETAILED DESCRIPTION OF THE INVENTION

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

For purposes of the present invention, an “organic halide” (alsosometimes referred to as a halogenated organic compound) denotes acompound which contains at least one carbon atom (or optionally sulfuror selenium atom, such as in the case of SF₆ and SeF6) and at least onehalogen atom selected from the group consisting of fluorine, chlorine,bromine, or iodine. Preferably the halogen is fluorine (i.e., thecompound is a fluorinated compound).

Most preferably the organic halide is a fluorinated compound which isperfluorinated (that is, fully fluorinated, e.g., a carbon compoundwherein all hydrogen atoms directly attached to the carbon atoms havebeen replaced by fluorine atoms). The perfluorinated organic halide(perfluorinated compound) is preferably a perfluorocarbon or aperfluoroether. The organic halide may be in the form of a gas, a liquid(including a gaseous precursor), or a solid. Preferably the organichalide is a liquid, even more preferably a liquid which is a gaseousprecursor that converts to a gas upon administration. Most preferably,the gaseous precursor converts to a gas at the site of (in close ortouching proximity to) the cell.

“Gaseous precursor” refers to a liquid or solid which is activated uponattaining a certain temperature or pressure to convert to a gas. Agaseous precursor which is capable of converting to a gas at the site ofthe cell may increase the efficiency of cellular uptake of compounds,and is therefore preferred.

Ideally, the gaseous precursors are liquid (or solid) at ambient (room)temperature (e.g., 25° C.), but will convert to a gas either atphysiological temperature (e.g., 37° C.) such as upon administration toa patient, or otherwise conveniently at the site of the cell such asupon application of heat (such as, for example, using ultrasound). Ifheat is applied, it should be done so at a temperature sufficient toconvert the gaseous precursor to a gas, but insufficient to harm thecell (e.g., denature the proteins, etc.). Thus, ideally a gaseousprecursor becomes a gas at less than about 80° C. Even more ideally, thegaseous precursor becomes a gas at between about 30° C. and about 70° C.Most ideally, the gaseous precursor becomes a gas at between about 37°C. and less than about 50° C.

A variety of different organic halides may be employed in thisinvention. Where the organic halide is a carbon based halide compound,the organic halide preferably contains from 1 to about 30 carbon atoms,more preferably 1 to about 24 carbon atoms, even more preferably 1 toabout 12 carbon atoms, still even more preferably about 5 to about 12carbon atoms, and most preferably about 6 to about 10 carbon atoms.Thus, the number of carbon atoms in the organic halide may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, carbon atoms, and upwards. Sulfur or selenium based halidecompounds, such as sulfur hexafluoride and selenium hexafluoride, arealso within the scope of the invention and the phrase organic halide asused herein. The organic halides contemplated herein may also, forexample, have carbon atoms interrupted by one or more heteroatoms, suchas —O— bonds (as in ether compounds) or have other substituents such asamines, etc. Preferred organic halides of the present invention are theperfluorinated organic halides such as perfluorocarbons andperfluoroethers.

Table 1 lists representative organic halides useful in the presentinvention. Other organic halides suitable for use in the presentinvention will be readily apparent to one skilled in the art, once armedwith the present disclosure. All such organic halides are intended tofall within the scope of the term organic halide, as used herein. TABLE1 Organic Halides Compound Boiling Point (° C.) 1. Mixed-halogenatedCompounds 1-bromo-nonafluorobutane 43 perfluorooctyliodide 160-161perfluoroocytlbromide 142 1-chloro-1-fluoro-1-bromomethane 381,1,1-trichloro-2,2,2-trifluoroethane 45.71,2-dichloro-2,2-difluoroethane 46 1,1-dichloro-1,2-difluoroethane 451,2-dichloro-1,1,3-trifluoropropane 50.4 1-bromoperfluorobutane 431-bromo-2,4-difluorobenzene 44 2-iodo-1,1,1-trifluoroethane 535-bromovaleryl chloride 43 1,3-dichlorotetrafluoroacetone 43 brominepentafluoride 40.3 1-bromo-1,1,2,3,3,3-hexafluoropropane 35.5 2-chloro1,1,1,4,4,4-hexafluoro-2-butene 33 2-chloropentafluoro-1,3-butadiene 37iodotrifluoroethylene 30 1,1,2-trifluoro-2-chloroethane 301,2-difluorochloroethane 35.5 1,1-difluoro-2-chloroethane 35.11,1-dichlorofluoroethane 31.8 heptafluoro-2-iodopropane 39bromotrifluoroethane −57.8 chlorotrifluoromethane −81.5dichlorodifluoromethane −29.8 dibromofluoromethane 23chloropentafluoroethane −38.7 bromochlorodifluoromethane −4dichloro-1,1,2,2-tetrafluoroethane 3.1-3.6 2. Fluorinated Compounds1,1,1,3,3-pentafluoropentane 40 perfluorotributylamine 178perfluorotripropylamine 130 3-fluorobenzaldehyde 562-fluoro-5-nitrotoluene 53 3-fluorostyrene 40 3,5-difluoroaniline 402,2,2-trifluoroethylacrylate 45 3-(trifluoromethoxy)-acetophenone 491,1,2,2,3,3,4,4-octafluorobutane 44.8 1,1,1,3,3-pentafluorobutane 401-fluorobutane 32.5 1,1,2,2,3,3,4,4-octafluorobutane 44.81,1,1,3,3-pentafluorobutane 40 perfluoro-4 methylquinolizidine 149perfluoro-N-methyl-decahydroquinone 150-155perfluoro-N-methyl-decahydroisoquinone 150-155perfluoro-N-cyclohexyl-pyrrolidine 145-152 tetradecaperfluoroheptane 76dodecaperfluorocyclohexane 52 3. Perfluorinated Compounds a.Perfluorocarbons perfluoromethane −129 perfluoroethane −78.3perfluoropropane −36 perfluorobutane −2 perfluoropentane 29.5perfluorohexane 59-60 perfluoroheptane 81 perfluorooctane 102perfluorononane 125 perfluorodecane ˜143 perfluorododecane melting pt75-77 perfluoro-2-methyl-2-pentene 51 perfluorocyclohexane 52perfluorodecalin 142 perfluorododecalin — perfluoropropylene −28perfluorocyclobutane −6 perfluoro-2-butyne −25 perfluoro-2-butene 1.2perfluorobuta-1,3-diene 6 b. Perfluoroether Compoundsperfluorobutylethyl ether 60 bis(perfluoroisopropyl) ether 54bis(perfluoropropyl) ether 59 perfluorotetrahydropyran 34perfluoromethyl tetrahydrofuran 27 perfluoro t-butyl methyl ether 36perfluoro isobutyl methyl ether — perfluoro n-butyl methyl ether 35.4perfluoro isopropyl ethyl ether — perfluoro n-propyl ethyl ether 23.3perfluoro cyclobutyl methyl ether — perfluoro cyclopropyl ethyl ether —perfluoro isopropyl methyl ether 36 perfluoro n-propyl methyl ether —perflouro diethyl ether   3-4.5 perfluoro cyclopropyl methyl ether —perfluoro methyl ethyl ether −23 perfluoro dimethyl ether −59 c. Othersulfur hexafluoride m.p. −50.5, sublimes −63.8 selenium hexafluoridem.p. −34.6 sublimes −46.6

Preferred organic halides include 1-bromo-nonafluorobutane,1,1,1,3,3-pentafluoropentane, perfluorohexane, perfluorocyclohexane,1-bromo-1,1,2,3,3,3-hexafluoropropane, heptafluoro-2-iodopropane,1,1,2,2,3,3,4,4-octafluorobutane, 1-fluorobutane,tetradecaperfluoroheptane and dodecaperfluorocylclohexane. Particularlypreferred are perfluorohexane (especially n-perfluorohexane) andperfluorocyclohexane. A wide variety of other organic halides useful inthe present invention will be readily apparent to those of skill in theart once armed with the present disclosure. Suitable additional organichalides include those, for example, disclosed in Long, Jr. in U.S. Pat.Nos. 4,987,154, 4,927,623, and 4,865,836, the disclosures of each ofwhich are hereby incorporated herein by reference in their entirety.

The amount of organic halide employed in the present invention may vary,as one skilled in the art will recognize, once armed with the presentdisclosure, and may be dependent on such factors as the particularorganic halide employed, type and nature of the compound to bedelivered, the age, weight, cells or patient (animal) to be treated, theparticular diagnostic, therapeutic or other application intended(including the disease state, if any, to be treated). Typically loweramounts are used and then increased until the desired delivery effect isachieved. Representative amounts are set forth in the examples herein.Of course, higher or lower amounts may be employed, as will berecognized by the skilled artisan.

Methods of introducing compounds into a cell (also referred to variouslyherein as methods for delivering a compound into a cell, methods ofintracellular delivery, methods of promoting, effecting, facilitating orenhancing the uptake of a compound into a cell, and the like) include“transfection”, which refers to the introduction of genetic material,i.e., a nucleotide sequence (e.g., DNA or RNA) into a host cell.Transfection is also sometimes referred to as transformation. DNA (orRNA) which is new to the cell into which it is incorporated is typicallyreferred to as heterologous DNA (or RNA) or exogenous DNA (or RNA). Somebacterial species take up exogenous DNA and do not discriminate betweenuptake of DNA from a similar or same species or from a completelydifferent species or organism. Exogenous DNA may also be taken up bycells, but may or may not be incorporated into nuclear material in ahereditable manner. The objective of transfection of a host cell may beto effect expression of one or more carefully selected sequences.

“Expression” and “gene expression” refer to the transcription and/ortranslation of a nucleic acid sequence resulting in the production of anamino acid, peptide and/or protein. The nucleic acid sequence may or maynot be incorporated into the genetic material of the host cell. Forexample, the nucleic acid sequence may be incorporated into the genomeof a host cell or may simply be introduced into the cell withoutincorporation into the genome.

Gene expression, upon administration of the composition of the presentinvention, may be effected (obtained., promoted, facilitated orenhanced), and in fact may be enhanced in that the expression of thenucleic acid sequences as compared to conventional transfectiontechniques such as calcium phosphate precipitation, viral vectors,microinjection, shock wave such as for example lithotripsy, andelectroporation, may be increased. Methods of measuring enhanced geneexpression will be known to skilled artisans once armed with the presentdisclosure and include enzyme-linked immunosorbent assay (ELISA) as wellas methods disclosed in Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), the disclosures of which are herebyincorporated herein by reference in their entirety. Thus, as a result ofthe methods of the present invention, a product (e.g., a protein) may beproduced. In addition, the prevention of the production of a product(such as, as a result of an antisense sequence delivered into the cell)by the host cell may also result.

Without being bound by any theory of operation, it is believed thatdelivery of nucleic acid sequences and other compounds in accordancewith the methods of the present invention may induce a cell to take upthe compound to be delivered thereto. Included within the definition ofdelivery of a compound into a cell in accordance with the methods of thepresent invention are active and passive mechanisms of cellular uptake.Ion channels and other means of transport utilized by cells toincorporate extracellular materials, including compounds to be deliveredthereto, into the intracellular milieu are encompassed by the presentinvention.

“Nucleotide sequence and nucleic acid sequence” refer to single anddouble stranded DNA and RNA sequences, including and not limited tooligonucleotide sequences of about 100 kb to about 1,000,000 kb(including whole chromosomes), preferably of about 4 kb to about 6 kb,more preferably about 1,000 nucleotides in length, more preferably about500 nucleotides in length, more preferably about 250 nucleotides inlength, more preferably about 100 nucleotides in length, more preferablyabout 50 nucleotides in length, more preferably about 25 nucleotides inlength, more preferably about 10 nucleotides in length, even morepreferably about 3 to about 10 kbp in length. Embodied by the term“nucleotide sequence” are all or part of a gene, at least a portion of agene, a gene fragment, a sense sequence, an antisense sequence, anantigene nucleic acid, a phosphorothioate oligodeoxynucleotide, and analteration, deletion, mismatch, transition, transversion, mutation,conservative substitution, and homolog of a sequence. The phrase “atleast a portion of,” and “all or part of,” as used herein, means thatthe entire gene need not be represented by the sequence so long as theportion of the gene represented is effective to block or exhibit,depending on the type of sequence used, gene expression. The sequencesmay be incorporated into an expression vector such as, and not limitedto, a plasmid, phagemid, cosmid, yeast artificial chromosome (YAC),virus (e.g., adenovirus, vaccinia virus, retrovirus), and defectivevirus (also known as a “helper virus”). The nucleotide sequence may alsobe administered naked, that is without an expression vector.

“Cell” and “host cell” refer to prokaryotic cells and eukaryotic cells,including plant cells, animal cells, cells of unicellular organisms,cells of multicellular organisms, etc. Especially preferred are animalcells, more preferably mammalian cells and most especially human cells,including but not limited to living cells, tissues, and organs.Eukaryotic cells are cells of higher organisms in which genetic materialis enclosed by a nuclear membrane. Prokaryotic cells are cells of lowerorganisms that lack a well defined nucleus and contain genetic materialthat is not enclosed within a membrane of its own. The cells may bepresent in vivo or in vitro (e.g. in cell culture).

The invention has wide applications for effecting (obtaining, promoting,facilitating or enhancing) and/or increasing the efficiency of,intracellular delivery (e.g., transfection) and/or, in the case ofnucleotides, gene expression in both in vitro and in vivo applications,and is particularly useful for prokaryotic and eukaryotic animal cells,particularly mammalian cells. Intracellular delivery includes deliveryinto the cells through a cell membrane (plasma membrane), cell wall,and/or nuclear membrane.

The phrase “cell membrane” (also termed “plasma membrane”) is used inits conventional sense as denoting the outer layer or boundary of thecytoplasm of a living cell. Cell membranes are typically comprised ofprotein and lipids, and are generally found in animal cells.

The phrase “cell wall” is also used in its conventional sense to denotea rigid or semi-rigid outer covering surrounding the protoplasts ofplant cells and most prokaryotes. Cell walls are typically found, forexample, in cells of bacteria, plants, algae, and fungi. Cell walls are,on the other hand, generally not present in animal cells. In plants, thewall typically comprises several layers; a primary wall composed ofcellulose microfibrils running through a matrix of hemicelluloses andpectic substances surrounded by a secondary wall composed of cellulosewhich is generally lignified to a varying extent. Cell walls of fungimay contain varying amounts of chitin. Cell walls of prokaryotes aretypically strengthened by mucopeptides and may be surrounded by amucilagenous capsule.

A wide variety of compounds can comprise the compounds to be deliveredto the cells in accordance with the invention, including bioactiveagents, diagnostic agents, pharmaceutical agents, and the like, andinclude proteins, DNA and RNA (both single and double-stranded),anti-sense and gene constructs, as well as other organic or inorganiccompounds. Whole genes, multiple gene sequences, and gene fragments maybe utilized as well as whole chromosomes and chromosome fragments.

As noted above, the methods of the present invention may, for example,be carried out in the presence of an organic halide, with or without theapplication of ultrasound, or in the absence of an organic halide butwith the application of ultrasound. Where bioactive agents other thannucleotides are employed as the compound to be delivered, generally, forbest results, an organic halide is used, although use of an organichalide in such a situation is not required.

If desired, the composition may further comprise a carrier. The carrieremployed may comprise a wide variety of materials. Carriers may include,for example, lipids, polymers, proteins, surfactants, inorganiccompounds, metal ions, and the like, alone or in combination with waterand/or a solvent, or the carrier may simply comprise water and/or asolvent. The lipids, proteins, and polymers, for example, may be inliquid form or solid form (such as, for example, the form of particles,fibers, sheets, layers, etc.), or may take the form of a vesicle orother stable, organized form, which may include but is not limited to,such forms commonly referred to as, for example, liposomes, micelles,bubbles, microbubbles, microspheres, lipid-, polymer-, and/orprotein-coated bubbles, microbubbles and/or microspheres, microballoons,aerogels, hydrogels, clathrates, hexagonal HII phase structures, and thelike. The internal void of the vesicle or other stable form may, forexample, be filled with a liquid (including, for example, a gaseousprecursor), a gas, a solid, or solute material, or any combinationthereof, including, for example, the compound to be delivered, theorganic halide, and/or any targeting ligand, as desired. Typically, thecarrier is provided as an aqueous milieu, such as water, saline (such asphosphate buffered saline), and the like, with or without other carriercomponents, although other non-aqueous solvents may also be employed, ifdesired. The carrier may comprise a mixture in the form of an emulsion,suspension, dispersion, solution, and the like. Lipid (including oil) inwater emulsions are especially preferred. As indicated above, thecarrier may also include buffers.

Thus, “vesicle”, as used herein, refers to an entity which is generallycharacterized by the presence of one or more walls or membranes whichform one or more internal voids. Vesicles may be formulated, forexample, from stabilizing compounds, such as a lipid, including thevarious lipids described herein, a polymer, including the variouspolymers described herein, or a protein, including the various proteinsdescribed herein, as well as using other materials that will be readilyapparent to one skilled in the art. Other suitable materials include,for example, any of a wide variety of surfactants, inorganic compounds,and other compounds as will be readily apparent to one skilled in theart. Also, as will be apparent to one skilled in the art upon readingthe present disclosure, the organic halides may themselves act assuitable carriers, and may in certain embodiments themselves formvesicles and other organized structures. Thus the use of the organichalides of the invention in combination with a compound to be delivered,without an additional compound to serve as a carrier, is within thescope of the invention. The lipids, polymers, proteins, surfactants,inorganic compounds, and/or other compounds may be natural, synthetic orsemi-synthetic. Preferred vesicles are those which comprise walls ormembranes formulated from lipids. The walls or membranes may beconcentric or otherwise. In the preferred vesicles, the stabilizingcompounds may be in the form of a monolayer or bilayer, and the mono- orbilayer stabilizing compounds may be used to form one or more mono- orbilayers. In the case of more than one mono- or bilayer, the mono- orbilayers may be concentric, if desired. Stabilizing compounds may beused to form unilamellar vesicles (comprised of one monolayer orbilayer), oligolamellar vesicles (comprised of about two or about threemonolayers or bilayers) or multilamellar vesicles (comprised of morethan about three monolayers or bilayers). The walls or membranes ofvesicles prepared from lipids, polymers or proteins may be substantiallysolid (uniform), or they may be porous or semi-porous. The vesiclesdescribed herein include such entities commonly referred to as, forexample, liposomes, micelles, bubbles, microbubbles, microspheres,lipid-, protein- and/or polymer-coated bubbles, microbubbles and/ormicrospheres, microballoons, microcapsules, aerogels, clathrate boundvesicles, hexagonal H II phase structures, and the like. The vesiclesmay also comprise a targeting ligand, if desired.

“Lipid vesicle”, “polymer vesicle” and “protein vesicle” referrespectively to vesicles formulated from one or more lipids, polymersand proteins.

“Liposome” refers to a generally spherical or spheroidal cluster oraggregate of amphipathic compounds, including lipid compounds, typicallyin the form of one or more concentric layers, for example, monolayers orbilayers. They may also be referred to herein as lipid vesicles. Theliposomes may be formulated, for example, from ionic lipids and/ornon-ionic lipids. Liposomes which are formulated from non-ionic lipidsmay also be referred to as “niosomes.”

“Micelle” refers to colloidal entities formulated from lipids. Incertain preferred embodiments, the micelles comprise a monolayer orhexagonal H2 phase configuration. In other preferred embodiments, themicelles may comprise a bilayer configuration.

“Aerogel” refers to generally spherical or spheroidal entities which arecharacterized by a plurality of small internal voids. The aerogels maybe formulated from synthetic or semisynthetic materials (for example, afoam prepared from baking resorcinol and formaldehyde), as well asnatural materials, such as polysaccharides or proteins.

“Clathrate” refers to a solid, semi-porous or porous particle which maybe associated with vesicles. In preferred form, the clathrates may forma cage-like structure containing cavities which comprise the vesicles.One or more vesicles may be bound to the clathrate. A stabilizingmaterial may, if desired, be associated with the clathrate to promotethe association of the vesicle with the clathrate. Suitable materialsfrom which clathrates may be formulated include, for example, porousapatites, such as calcium hydroxyapatite, and precipitates of polymersand metal ions, such as alginic acid precipitated with calcium salts.

“Emulsion” refers to a mixture of two or more generally immiscibleliquids and is generally in the form of a colloid. The liquids may behomogeneously or heterogeneously dispersed throughout the emulsion.Alternatively, the liquids may be aggregated in the form of, forexample, clusters or layers, including mono- or bilayers.

“Suspension” or “dispersion” refers to a mixture, preferably finelydivided, of two or more phases (solid, liquid or gas), such as, forexample, liquid in liquid, solid in liquid, liquid in gas, etc.) whichcan preferably remain stable for extended periods of time.

“Hexagonal H II phase structure” refers to a generally tubularaggregation of lipids, proteins, or polymers (especially lipids) inliquid media, for example, aqueous media, in which any hydrophilicportion(s) generally face inwardly in association with an aqueous liquidenvironment inside the tube. The hydrophobic portion(s) generallyradiate outwardly and the complex assumes the shape of a hexagonal tube.A plurality of tubes is generally packed together in the hexagonal phasestructure.

“Biocompatible” refers to materials which are generally not injurious tobiological functions and which will not result in any degree ofunacceptable toxicity, including allergenic responses and diseasestates. The compositions of the present invention and/or componentsthereof are typically biocompatible.

The nucleotide sequence or other compound to be delivered may beadministered, if desired, “in combination with” an organic halide, andmay further be administered, if desired, “in combination with” acarrier, including a vesicle (or other stable form). “In combinationwith” refers to the co-administration of the compound to be deliveredand the organic halide (and/or carrier, if desired). The compound to bedelivered and the organic halide (and/or any carrier) may be combined inany of a variety of different fashions, including simply being placed inadmixture with one another. In addition, for example, the nucleotide orother compound to be delivered and/or the organic halide may beembedded, encapsulated, or attached to, or with, one another, as desired(including any and all combinations thereof). The phrase “in admixture”includes solutions, suspensions, emulsions, dispersions, mixtures, etc.The phrase “attached to” or variations thereof, as used herein, denotesbeing linked in some manner, such as through a covalent or ionic bond orother means of chemical or electrochemical linkage or interaction. Thephrase “encapsulated” and variations thereof as used herein refers to alocation inside an internal void of a vesicle or other structure. Thephrase “embedded within” or variations thereof as used herein signifiesa positioning within the wall of a vesicle or other structure. Thus, anucleotide sequence, for example, can be positioned variably, such as,for example, entrapped within the internal void of the vesicle, situatedon the internal wall of the vesicle, incorporated onto the externalsurface of the vesicle, and/or enmeshed within the vesicle structureitself. In addition, one or more vesicles may be administered as acavitator. In such case, the vesicles accompany the administration of acompound and may serve to enhance the efficiency of ultrasound.

Lipids may be used in the present invention as a carrier. The lipids maybe natural, synthetic or semisynthetic (i.e., modified natural). Lipidsuseful in the invention include, and are not limited to, fatty acids,lysolipids, oils (including safflower, soybean and peanut oil),phosphatidylcholine with both saturated and unsaturated lipids includingphosphatidylcholine; dioleoylphosphatidylcholine;dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine,dilauroylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine; distearoylphosphatidylcholine;phosphatidylethanolamines such as dioleoylphosphatidylethanolamine;phosphatidylserine; phosphatidylglycerol; phosphatidylinositol,sphingolipids such as sphingomyelin; glycolipids such as ganglioside GM1and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acid;palmitic acid; stearic acid; arachidonic acid; oleic acid; lipidsbearing polymers such as polyethyleneglycol, chitin, hyaluronic acid orpolyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate and cholesterolhemisuccinate; tocopherol hemisuccinate, lipids with ether andester-linked fatty acids, polymerized lipids (a wide variety of whichare known in the art), diacetyl phosphate, stearylamine, cardiolipin,phospholipids with short chain fatty acids of about 6 to about 8 carbonsin length, synthetic phospholipids with asymmetric acyl chains (e.g.,with one acyl chain of about 6 carbons and another acyl chain of about12 carbons), 6-(5-cholesten-3b-yloxy)-1-thio-β-D-galactopyranoside,digalactosyldiglyceride,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside,12-(((7′-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoicacid; N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalmitic acid;(cholesteryl)₄′-trimethyl-ammonio)butanoate;N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinyl-glycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoyl-glycerophosphoethanolamine andpalmitoylhomocysteine, and/or combinations thereof. Vesicles or otherstructures may be formed of the lipids, either as monolayers, bilayers,or multilayers and may or may not have a further coating.

The preferred lipid carrier may be in the form of a monolayer orbilayer, and the mono- or bilayer may be used to form one or more mono-or bilayers. In the case of more than one mono- or bilayer, the mono- orbilayers may be concentric. The carrier may form a unilamellar vesicle(comprised of one monolayer or bilayer), an oligolamellar vesicle(comprised of about two or about three monolayers or bilayers) or amultilamellar vesicle (comprised of more than about three monolayers orbilayers). The walls or membranes of a vesicle may be substantiallysolid (uniform), or they may be porous or semi-porous.

Lipids bearing hydrophilic polymers such as polyethyleneglycol (PEG),including and not limited to PEG 2,000 MW, 5,000 MW, and PEG 8,000 MW,are particularly useful for improving the stability and sizedistribution of organic halide-containing composition.Dipalmitoylphosphatidylcholine (DPPC) may be useful in the presentinvention at about 70% to about 90%,dipalmitoylphosphatidylethanolamine-polyethylene glycol 5000 (DPPE-PEG5000) may be useful at about 0% to about 20% and dipalmitoylphosphatidicacid (DPPA) may be useful at about 0% to about 20% (all percentagesbeing in mole percent molecular weight). A preferred product which ishighly useful as a carrier in the present invention contains about 82mole percent DPPC, about 8 mole percent DPPE-PEG 5,000 MW and about 10mole percent DPPA. Various different mole ratios of PEGylated lipid arealso useful.

Additionally lipid moieties capable of polymerization are embraced inthe invention as coatings for the vesicles. Examples of these include,but are not limited to, alkenyl and alkynyl moieties, such as oleyl andlinoleyl groups, diacetylene, acryloyl and methacryloyl groups with orwithout polar groups to enhance water solubility, cyanoacrylate estersoptionally carrying lipophilic esterifying groups or the compoundsillustrated as A and B, below. A number of such compounds are described,for example, in Klaveness et al., U.S. Pat. No. 5,536,490. Thedisclosures of Klaveness et al., U.S. Pat. No. 5,536,490, are herebyincorporated herein by reference in their entirety.

Fluorinated or perfluorinated lipids may also be used in this invention,either as the organic halide component or as an additional carriermaterial. Examples of suitable fluorinated lipids include but are notlimited to compounds of the formulaC_(n)F_(2n+1)(CH₂)_(m)C(O)OOP(OO⁻)O(CH₂)_(w)N⁺(CH₃)₃C_(n)F_(2n+1)(CH₂)_(m)C(O)Owherein: m is 0 to about 18, n is 1 to about 12; and w is 1 to about 8.Examples of and methods for the synthesis of these, as well as otherfluorinated lipids useful in the present invention, are set forth inU.S. application Ser. No. 08/465,868, filed Jun. 6, 1995, Reiss et al.U.S. Pat. No. 5,344,930, Frezard, F., et al., Biochem Biophys Acta 1994,1192:61-70, and Frezard, F., et al., Art. Cells Blood Subs and ImmobBiotech. 1994, 22:1403-1408, the disclosures of each of which areincorporated herein by reference in their entirety. One specific exampleof a difluoroacyl glycerylphosphatidylcholine, nonafluorinated diacylglycerylphosphatidylcholine, is represented by compound A, below. Thoseskilled in the art will appreciate that analogous fluorinatedderivatives of other common phospholipids (diacylphosphatidyl serine,diacylphosphatidyl ethanolamine, diacylphosphatidyl glycerol,diacylphosphatidyl glycerol, etc.) as well as fluorinated derivatives offatty acyl esters and free fatty acids may also function in accordancewith the scope of the invention.

Additionally lipid based and fluorinated (including perfluorinated)surfactants such as may be used as carriers in the present invention.

A wide variety of such fluorinated compounds may be employed, including,for example, the class of compounds which are commercially available asZONYL® fluorosurfactants (the DuPont Company, Wilmington, Del.),including the ZONYL® phosphate salts and ZONYL® sulfate salts, which arefluorosurfactants having terminal phosphate or sulfate groups.Representative compounds are disclosed, for example, in U.S. Pat. No.5,276,145, the disclosures of which are hereby incorporated herein byreference in their entirety. Suitable ZONYL® surfactants also include,for example, ZONYL® surfactants identified as Telomer B, includingTelomer B surfactants which are pegylated (i.e., have at least onepolyethylene glycol group attached thereto), also known as PEG-TelomerB, available from the DuPont Company. Most preferred are such pegylatedfluorosurfactants.

Suitable polymerizable and/or fluorinated compounds include

In formula A, above, preferably x is an integer from about 8 to about18, and n is 2×. Most preferably x is 12 and n is 24.

Cationic lipids and other derivatized lipids and lipid mixtures also maybe useful as carriers in the present invention. Suitable cationic lipidsinclude dimyristyl oxypropyl-3-dimethylhydroxy ethylammonium bromide(DMRIE), dilauryl oxypropyl-3-dimethylhydroxy ethylammonium bromide(DLRIE), N-[1-(2,3-dioleoyloxyl)propal]-n,n,n-trimethylammonium sulfate(DOTAP), dioleoylphosphatidylethanolamine (DOPE),dipalmitoylethylphosphatidylcholine (DPEPC), dioleoylphosphatidylcholine(DOPC), polylysine, lipopolylysine, didoceyl methylammonium bromide(DDAB),2,3-dioleoyloxy-N-[2-(sperninecarboxamidoethyl]-N,N-di-methyl-1-propanaminiumtrifluoroacetate (DOSPA), cetyltrimethylammonium bromide (CTAB),lysyl-PE, 3,β-[N,(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Cholesterol, also known as DC-Chol), (-alanyl cholesterol,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),dipalmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES),dicaproylphosphatidylethanolamine (DCPE), 4-dimethylaminopyridine(DMAP), dimyristoylphosphatidylethanolamine (DMPE),dioleoylethylphosphocholine (DOEPC), dioctadecylamidoglycyl spermidine(DOGS),N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,N-dimethylammoniumiodide (DOHME), Lipofectin (DOTMA+DOPE, Life Technologies, Inc.,Gaithersburg, Md.), Lipofectamine (DOSPA+DOPE, Life Technologies, Inc.,Gaithersburg, Md.), Transfectace (Life Technologies, Inc., Gaithersburg,Md.), Transfectam (Promega Ltd., Madison, Wis.), Cytofectin (LifeTechnologies Inc., Gaithersburg, Md.). Other representative cationiclipids include but are not limited to phosphatidylethanolamine,phospatidylcholine, glycero-3-ethylphosphatidylcholine and fatty acylesters thereof, di- and trimethyl ammonium propane, di- andtri-ethylammonium propane and fatty acyl esters thereof. A preferredderivative from this group isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).Additionally, a wide array of synthetic cationic lipids function ascompounds useful in the invention. These include common natural lipidsderivatized to contain one or more basic functional groups. Examples oflipids which may be so modified include but are not limited todimethyldioctadecylammonium bromide, sphingolipids, sphingomyelin,lysolipids, glycolipids such as ganglioside GM1, sulfatides,glycosphingolipids, cholesterol and cholesterol esters and salts,N-succinyldioleoylphosphatidylethanolamine, 1,2,-dioleoyl-sn-glycerol,1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphatidyl-ethanolamine andpalmitoylhomocystiene.

Other synthesized cationic lipids that are useful in the presentinvention are those disclosed in pending U.S. patent application Ser.No. 08/391,938, filed Feb. 2, 1995, and include, for example, N,N′-Bis(dodecyaminocarbonylmethylene)-N,N′bis((-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)ethylenediaminetetraiodide; N,N″-Bis(hexadecylaminocarbonylmethylene)-N,N′,N″-tris((-N,N,N-trimethylammonium-ethylaminocarbonylmethylenedi-ethylenetriaminehexaiodide; N,N′-Bis(dodecylaminocarbonylmethylene)-N,N′-bis((-N,N,N-trimethylammoniumethylamino-carbonylmethylene)cyclohexylene-1,4-diaminetetraiodide;1,1,7,7-tetra-((-N,N,N,N-tetramethylammoniumethylaminocarbonylmethylene)-3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptaneheptaiodide; andN,N,N′N′-tetra((-N,N,N-trimethylammoniumethylaminocarbonylmethylene)-N′-(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetriaminetetraiodide. Those of skill in the art will recognize that countlessother natural and synthetic variants carrying positive charged moietieswill also function in the invention.

Also useful as carriers in the present invention are a wide variety ofsurfactants (i.e., surface-active agents), including polyoxyalkylenefatty acid esters (such as polyoxyethylene fatty acid esters),polyoxyalkylene fatty alcohols (such as polyoxyethylene fatty alcohols),polyoxyalkylene fatty alcohol ethers (such as polyoxyethylene fattyalcohol ethers), polyoxyalkylene sorbitan fatty esters (such as, forexample, the class of compounds referred to as TWEEN™, commerciallyavailable from ICI Americas, Inc., Wilmington, Del.), includingpoly(oxyethylene)poly(oxypropylene) copolymers (such as Pluronics),polysorbates (such as Tween20, Tween40, and Tween80), polyoxyethylenealcohols (such as Brij), and plasmalogens, the term applied to a numberof a group of phospholipids present in platelets that liberate higherfatty aldehydes, e.g. palmital, on hydrolysis and may be related to thespecialized function of platelets in blood coagulation and plasmalogensare also present in cell membranes of muscle and the myelin sheath ofnerve fibers.

In the preferred embodiment of the invention the organic halide isincorporated into the core of a vesicle which vesicle carrier is alsoused to complex the compound to be delivered, such as DNA.

A wide variety of oils may be preferably employed as carriers in thepresent invention including, but not limited to, safflower, soybean, andpeanut oil. The composition may take the form of an oil in wateremulsion if desired.

The most preferred carrier is a cationic lipid (including a cationicliposome), particularly as employed in an aqueous milieu. A preferredcationic lipid is DPEPC in admixture with the neutral fusogenic lipiddioleoylphosphatidylethanolamine (DOPE). A preferred ratio of lipid toorganic halide is 5:1 w/w. A preferred embodiment is to formulate thelipid or polymer as an organic halide-filled microsphere, such as amicrosphere formed with the lipids dipalmitoylphosphatidylcholine(DPPC), dipalmitoylphosphatidylethanolamine coupled to polyethyleneglycol 5000 (DPPE-PEG5000), and dipaInitoylphosphatidic acid (DPPA).DPPC:DPPE-PEG5000:DPPA may be combined in a ratio of about 82%:8%:10%(mole %) or 83%:8%:5%. DPPE-PEG5000 is comprised of DPPE and PEG5000 ina ratio of about 20%:80% (weight %). PEG5000 refers to PEG having anaverage molecular weight of about 5000.

Proteins (including peptides) useful as carriers in accordance with thepresent invention include molecules comprising, and preferablyconsisting essentially of, α-amino acids in peptide linkages. A widevariety of proteins may be employed as carriers in the presentinvention, including natural, synthetic, or semi-synthetic proteins.Included within the term “protein” are globular proteins, such asalbumins, globulins and histones, and fibrous proteins such ascollagens, elastins and keratins. Also included are “compound proteins”,wherein a protein molecule is united with a nonprotein molecule, such asnucleproteins, mucoproteins, lipoproteins, and metalloproteins.Preferable proteinaceous macromolecules include for example, albumin,collagen, polyarginine, polylysine, polyhistidine, γ-globulin andβ-globulin, with albumin, polyarginine, polylysine, and polyhistidinebeing more preferred. Fluorinated peptides and synthetic pseudopeptidesare also useful as carriers. Fluorinated peptides useful in the presentinvention include those described in Lohrmann, U.S. Pat. No. 5,562,892,the disclosures of which are hereby incorporated herein by reference intheir entirety. Cationic peptides may also be usefully employed ascarriers in the present invention. Various peptides suitable for use inthe present invention will be apparent to one skilled in the art basedon the present disclosure.

The methods of the present invention may also involve vesicles or otherorganized stable form formulated from proteins, peptides and/orderivatives thereof. Vesicles which are formulated from proteins andwhich would be suitable for use in the methods of the present inventionare described, for example, in Feinstein, U.S. Pat. Nos. 4,572,203,4,718,433, and 4,774,958, and Cerny et al., U.S. Pat. No. 4,957,656, allof the disclosures of each of which are hereby incorporated by referencein their entirety. Other protein-based vesicles, in addition to thosedescribed in the aforementioned patents, would be apparent to one ofordinary skill in the art, once armed with the present disclosure.

Included among the methods described in the aforementioned patents forthe preparation of protein-based vesicles are methods which involvesonicating a solution of a protein. In preferred form, the startingmaterial may be an aqueous solution of a heat-denaturable, water-solublebiocompatible protein. The encapsulating protein is preferablyheat-sensitive so that it can be partially insolubilized by heatingduring sonication. Suitable heat-sensitive proteins include, forexample, albumin, hemoglobin, collagen, and the like. Preferably, theprotein is a human protein, with human serum albumin (HSA) being morepreferred. HSA is available commercially as a sterile 5% aqueoussolution, which is suitable for use in the preparation of protein-basedvesicles. Of course, as would be apparent to one of ordinary skill inthe art, other concentrations of albumin, as well as other proteinswhich are heat-denaturable, can be used to prepare the vesicles.Generally speaking, the concentration of HSA can vary and may range fromabout 0.1 to about 25% by weight, and all combinations andsubcombinations of ranges therein. It may be preferable, in connectionwith certain methods for the preparation of protein-based vesicles, toutilize the protein in the form of a dilute aqueous solution. Foralbumin, it may be preferred to utilize an aqueous solution containingfrom about 0.5 to about 7.5% by weight albumin, with concentrations ofless than about 5% by weight being preferred, for example, from about0.5 to about 3% by weight.

The protein-based vesicles may be prepared using equipment which iscommercially available. For example, in connection with a feedpreparation operation as disclosed, for example, in Cerny, et al., U.S.Pat. No. 4,957,656, stainless steel tanks which are commerciallyavailable from Walker Stainless Equipment Co. (New Lisbon, Wis.), andprocess filters which are commercially available from Millipore(Bedford, Mass.), may be utilized.

The sonication operation may utilize both a heat exchanger and a flowthrough sonicating vessel, in series. Heat exchanger equipment of thistype may be obtained from ITT Standard (Buffalo, N.Y.). The heatexchanger maintains operating temperature for the sonication process,with temperature controls ranging from about 65° C. to about 80° C.,depending on the makeup of the media. The vibration frequency of thesonication equipment may vary over a wide range, for example, from about5 to about 40 kilohertz (kHz), with a majority of the commerciallyavailable sonicators operating at about 10 or 20 kHz. Suitablesonicating equipment include, for example, a Sonics & MaterialsVibra-Cell, equipped with a flat-tipped sonicator horn, commerciallyavailable from Sonics & Materials, Inc. (Danbury, Conn.). The powerapplied to the sonicator horn can be varied over power settings scaledfrom 1 to 10 by the manufacturer, as with Sonics & Materials Vibra-CellModel VL1500. An intermediate power setting, for example, from 5 to 9,can be used. It is preferred that the vibrational frequency and thepower supplied be sufficient to produce cavitation in the liquid beingsonicated. Feed flow rates may range from about 50 mL/min to about 1000mL/min, and all combinations and subcombinations of ranges therein.Residence times in the sonication vessel can range from about 1 secondto about 4 minutes, and gaseous fluid addition rates may range fromabout 10 cubic centimeters (cc) per minute to about 100 cc/min, or 5% to25% of the feed flow rate, and all combinations and subcombinations ofranges therein.

It may be preferable to carry out the sonication in such a manner toproduce foaming, and especially intense foaming, of the solution.Generally speaking, intense foaming and aerosolating are important forobtaining a contrast agent having enhanced concentration and stability.To promote foaming, the power input to the sonicator horn may beincreased, and the process may be operated under mild pressure, forexample, about 1 to about 5 psi. Foaming may be easily detected by thecloudy appearance of the solution, and by the foam produced.

Such sonication methods may also be employed to prepare lipid-based orother types of carriers as will be apparent to the skilled artisan.

Suitable methods for the preparation of protein-based vesicles mayinvolve physically or chemically altering the protein or proteinderivative in aqueous solution to denature or fix the material. Forexample, protein-based vesicles may be prepared from a 5% aqueoussolution of HSA by heating after formation or during formation of thecontrast agent via sonication. Chemical alteration may involvechemically denaturing or fixing by binding the protein with adifunctional aldehyde, such as glutaraldehyde. For example, the vesiclesmay be reacted with 0.25 grams of 50% aqueous glutaraldehyde per gram ofprotein at pH 4.5 for 6 hours. The unreacted glutaraldehyde may then bewashed away from the protein.

The carriers may also be formulated with polymers, natural, synthetic,or semisynthetic. A wide variety of polymers may be utilized as carriersin the present invention, including synthetic polymers includingpolyethylenes (such as, for example, polyethylene glycol),polyoxyethylenes (such as, for example, polyoxyethylene glycol),polypropylenes (such as, for example, polypropylene glycol), pluronicacids and alcohols, polyvinyls (such as, for example, polyvinylalcohol), and polyvinylpyrrolidone. Exemplary natural polymers suitablefor use in the present invention include polysaccharides.Polysaccharides include, for example, arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans (such as, forexample, inulin), levan, fucoidan, carrageenan, galactocarolose, pectin(including high methoxy pectin and low methoxy pectin; with low methoxypectin denoting pectin in which less than 40% of the carboxylic acidgroups are esterified and/or amidated, and high methoxy pectin denotingpectin in which 40% or more of the carboxylic acid groups are esterifiedand/or amidated), pectic acid, amylose, pullulan, glycogen, amylopectin,cellulose, carboxylmethylcellulose, hydroxypropyl methylcellulose,dextran, pustulan, chitin, agarose, keratan, chondroitin, dermatan,hyaluronic acid and alginic acid, and various other homopolymers orheteropolymers such as those containing one or more of the followingaldoses, ketoses, acids or amines: erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose,idose, galactose, talose, erythrulose, ribulose, xylulose, psicose,fructose, sorbose, tagatose, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, guluronic acid, glucosamine,galactosamine and neuraminic acid. It is recognized that some polymersmay be prepared by chemically modifying naturally occurring polymers.Such chemically modified natural polymers also referred to assemisynthetic polymers. The polymers employed may also comprisefluorinated polymers, including those described in Lohrmann, U.S. Pat.No. 5,562,892, the disclosures of which are hereby incorporated hereinby reference in their entirety. Furthermore, the polymers may be in theform of vesicles, such as for example, those described in Unger, U.S.Pat. No. 5,205,290, the disclosures of which are hereby incorporatedherein by reference in their entirety. As used herein, the term“polymer” denotes molecules formed from the chemical union of two ormore repeating units, and include dimers, trimers, and oligomers. Inpreferred form, the term “polymer” refers to molecules which comprise 10or more repeating units.

Metal ions may also be employed as carriers in the present invention.Suitable metal ions include calcium ions, magnesium ions, zinc ions, andthe like, as well as a wide variety of inorganic compounds. Othersuitable metal ions as well as other suitable inorganic compounds willbe readily apparent to those skilled in the art once armed with thepresent invention.

Other useful agents that may be employed in the carrier of the presentinvention include osmotic agents, anti-microbials, viscosity raisingagents, suspending agents, humectants and anti-humectants, dependingupon the particular formulation desired.

One or more emulsifying or stabilizing agents may also be employed as orbe included in the carrier. These agents help to maintain the size ofany discrete units (e.g., liquid droplets, particles, gas bubbles, etc.)of the organic halide and/or compounds to be delivered that may haveformed the composition. The size of these discrete units will generallyaffect the size of any resultant gas bubbles that may form from anygaseous precursors. The emulsifying and stabilizing agents also may beused to generally coat or stabilize the organic halides, compounds to bedelivered, etc. Stabilization is desirable to maximize the intracellulardelivery effect. Although stabilization is preferred, this is not anabsolute requirement. Because any gas resulting from organic halidegaseous precursors is more stable than air, they may still be designedto provide useful delivery means; for example, they pass through thepulmonary circulation following peripheral venous injection, even whennot specifically stabilized by one or more coating or emulsifyingagents. One or more coating or stabilizing agents is preferred however,as are flexible stabilizing materials. Also, it should be noted thatcompositions stabilized by polysaccharides, gangliosides, and polymersare generally more effective than those stabilized by albumin and otherproteins. Also, liposomes prepared using aliphatic compounds arepreferred, since microspheres stabilized with these compounds are muchmore flexible and stable to pressure changes.

The carrier of the invention may also comprise a wide variety ofviscosity modifiers, including and not limited to carbohydrates andtheir phosphorylated and sulfonated derivatives; polyethers, preferablywith molecular weight ranges between 400 and 8000; di- and trihydroxyalkanes and their polymers, preferably with molecular weight rangesbetween 800 and 8000. Glycerol propylene glycol, polyethylene glycol,polyvinyl pyrrolidone, and polyvinyl alcohol may also be useful ascarriers or stabilizers in the present invention. Particles which areporous or semi-solid such as hydroxyapatite, metal oxides andcoprecipitates of gels, e.g., hyaluronic acid with calcium may be usedand may formulate a center or nidus to stabilize compositions of theinvention.

Emulsifying and/or solubilizing agents may also be used in a carrier,particularly in conjunction with lipids or liposomes. Such agentsinclude and are not limited to, acacia, cholesterol, diethanolamine,glyceryl monostearate, lanolin alcohols, lecithin, mono- anddi-glycerides, mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer,polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleylether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan, sorbitan mono-laurate, sorbitan mono-oleate,sorbitan mono-palmitate, sorbitan monostearate, stearic acid, trolamine,and emulsifying wax. All lipids with perfluoro fatty acids as acomponent of the lipid in lieu of the saturated or unsaturatedhydrocarbon fatty acids found in lipids of plant or animal origin may beused. Suspending and/or viscosity-increasing agents that may beparticularly useful with lipid or liposome solutions include but are notlimited to, acacia, agar, alginic acid, aluminum mono-stearate,bentonite, magma, carbomer 934P, carboxymethylcellulose, calcium andsodium and sodium 12, glycerol, carrageenan, cellulose, dextrin,gelatin, guar gum, hydroxyethyl cellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin,polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol,alginate, silicon dioxide, sodium alginate, tragacanth, and xanthum gum.A preferred product of the present invention incorporates lipid as amixed solvent system in a ratio of 8:1:1 or 9:1:1 normalsaline:glycerol:propylene glycol.

The amount of carrier material employed in connection with the subjectinvention may vary, as one skilled in the art will recognize upon beingplaced in possession of the subject disclosure, and may be dependent onsuch factors as the particular carrier used, the type and nature of thecompound to be delivered, the age, weight, cells or patient (animal) tobe treated, the particular diagnostic, therapeutic or other applicationintended (including the disease state, if any, to be treated), and theorganic halide (if any) used. Generally, smaller amounts of carrier areemployed, and increased until the desired delivery result is obtained.Representative amounts are set forth in the examples herein. Of course,higher or lower amounts may be employed, as will be recognized by theskilled artisan.

A wide variety of different methods may be used to mix the organichalide, compound to be delivered, and/or carrier, and incorporate thecompound to be delivered with or into any organic halide and/or carrier.Methods include shaking by hand, vortexing, mechanical shaking (e.g.with an Espe CapMix, Espe Medizin-Dental GMBH, Seefeld, Germany),extruder (e.g. with a Lipex Biomembranes Extruder Device, Vancouver,B.C., Canada), microemulsification (e.g. with a Microfluidizer,Microfluidics Corp., Newton, Mass.), mixing with static in line mixers(Cole-Parmer Instrument Co., Vernon Hills, Ill.), spray drying (e.g.with a Bucchi spray dryer, Brinkmann Ind., Inc., Westbury, Mass.),mechanical stirring/mixing (e.g. with a Silverson Mixer, SilversonMachines, Ltd., Waterside Chesham Bucks, England) and sonication. Ingeneral it is desirable to mix the carrier (e.g. lipids such as DPEPCand DOPE) together with the organic halide prior to adding the compoundto be delivered (e.g., DNA). After adding the DNA, a carrier and organichalide association will form with the DNA. If desired, additional mixingmay then be performed by one of the above techniques. In some othersituations, e.g. calcium precipitation, the DNA, organic halide, andcations may be added together with one or more stabilizing agents toform the precipitates of DNA/carrier/organic halide in a single stepprocess. Again, one of a variety of mixing techniques as described abovemay be employed to decrease the size of the resultant particles.

The carriers may be combined with the compound to be delivered and theorganic halide in varying amounts and percentages, as will be understoodby those skilled in the art once armed with the present disclosure.Typically, smaller amounts of all compositional components are employed,and increased selectively in increments until the desired deliveryeffect is achieved. Generally, when the compound to be delivered isemployed with a carrier, the ratio of organic halide and any carrier tothe compound to be delivered may be from about 6 to about 1, to about 1to about 6, and variations therebetween. Preferably, the carrier tocompound to be delivered ratio is about 6 to about 1. Representativeratios are provided by the examples herein. Of course, other ratios canbe suitably employed over a wide variety of ranges as desired, as willbe recognized by the skilled artisan, and all such ratios are within thescope of the present invention.

The resulting composition may be stored as a lyophilized, or freezedried, material for inhalation or hydration prior to use or as apreformed suspension. Cryopreservatives known to skilled artisans oncearmed with the present disclosure may be used in the lyophilized form ofthe composition. To prevent agglutination or fusion of vesicles as aresult of lyophilization, it may be useful to include additives whichprevent such fusion or agglutination from occurring. Additives which maybe useful include sorbitol, mannitol, sodium chloride, glucose,trehalose, polyvinylpyrrolidone and poly(ethylene glycol) (PEG), forexample, PEG polymers having a molecular weight of from about 400 toabout 10,000, with PEG polymers having molecular weights of about 1000,3000 (such as PEG3350) and 5000 being preferred. These and otheradditives are described in the literature, such as in the U.S.Pharmacopeia, USP XXII, NF XVII, The United States Pharmacopeia, TheNational Formulary, United States Pharmacopeial Convention Inc., 12601Twinbrook Parkway, Rockville, Md. 20852, the disclosures of which arehereby incorporated herein by reference in their entirety. Lyophilizedpreparations generally have the advantage of greater shelf life. Asnoted above, if desired, the lyophilized composition may be (andpreferably is) rehydrated prior to use.

The route of administration varies depending upon the intendedapplication. For cell culture applications, the composition is typicallycontacted with the cells by, for example, adding it to the cell culturemedia or applying it directly to the cells. Advantages of this inventionfor transfection in cell culture media include high activity in serumcontaining media and a single step transfection process with higherefficiency transfection than in other more complicated systems. Indeed,the present invention makes it possible to obtain gene expression incells in which transfection was otherwise impossible or extremelydifficult. For in vivo administration the composition may simply beinjected, such as intravenously, intravascularly, intralymphatically,parenterally, subcutaneously, intramuscularly, intranasally,intrarectally, intraperitoneally, interstitially, into the airways vianebulizer, hyperbarically, orally, topically, or intratumorly, orotherwise administered.

One or more targeting ligands may be incorporated into the carrier tofacilitate uptake by selected cells. Targeting ligands include, forexample, peptides, antibodies, antibody fragments, glycoproteins,carbohydrates, etc. Preferably, the targeting ligand is covalentlyattached to the carrier, e.g., to a lipid. Preferably the targetingligand is attached to a linker which is attached to the surface of thecarrier. Preferred linkers are polymers, for example, bifunctional PEGhaving a molecular weight of about 1,000 to about 10,000, mostpreferably 5,000. Generally, the targeting ligand is incorporated intothe carrier from about 0.1 mole % to about 25 mole %, preferably about 1mole % to about 10 mole %.

In this regard, the composition may be targeted to coated pits ofselected cells and taken up into endosomes via a process of receptormediated endocytosis. If desired ultrasound energy may be applied to thetarget tissue to facilitate gene expression. For inhalation thecomposition may be inhaled via a nebulizer or via an inhaler. Also, oralor rectal routes may be utilized to administer these composition.Transcutaneous application may be accomplished by the use of penetrationenhancing agents with or without the application of sonophoresis (e.g.low frequency sound in range of 10 to 100 Khz) or iontophoresis. Alsointerstitial (e.g. intratumoral) and subcutaneous injection may beperformed to administer the composition.

Also the invention may be practiced with gene gun techniques orelectroporation, or in combination with other transfection techniquesknown in the art. In either case, ultrasound may be applied to the cellsbefore, after, and/or simultaneously with the gene gun orelectroporation procedure. The electric fields of electroporation mayalso be pulsed in tandem with the ultrasound energy to further increasethe efficacy of transfection.

The compounds and compositions may, in accordance with the presentinvention, be administered alone, or together with ultrasound. Ifultrasound is employed, it is administered at a frequency and energylevel sufficient to assist in inducing the uptake of the compound to thecell. Where organic halide gaseous precursors are employed, theultrasound may be applied at a frequency and energy level sufficient toconvert the organic halide gaseous precursor to a gas. For example, thepresent invention of administering compounds to cells includesadministering a nucleotide sequence (or other compound of interest to bedelivered) to a cell and applying ultrasound to the cell for a timeeffective to induce the uptake of the nucleotide sequence (or othercompound). Enhanced delivery of the compound (and expression of thenucleotide sequence, in the case of nucleotide sequence beingadministered) results. Ultrasound is carried out at a frequency, energylevel, and duty cycle for a therapeutically effective time in which toinduce delivery of the nucleotide sequence. Suitable frequencies, energylevels and duty cycles are disclosed herein, and other ranges will bereadily apparent to one skilled in the art once armed with the presentdisclosure.

The methods of the present invention permit the delivery of sequencescoding for the gene expression of a variety of proteins, and antisensesequences which block gene expression of a variety of proteins. As aresult, a number of diseases may be treated with the transfectionmethods of the present invention. In addition, the methods oftransfection of the present invention may be practiced in vivo, ex vivo,and in vitro.

Administration of a nucleotide sequence by a microsphere utilizes anucleotide sequence attached to a microsphere in various positionsrelative to the microsphere as set forth above. While not intending tobe bound by any particular theory or theories of operation, themicrosphere approach is believed to rely on the fusion of the nucleotidesequence containing microsphere with the plasma membrane of the hostcell. The nucleotide sequence subsequently traverses the cytoplasm andenters the nucleus. The use of a microsphere results in little toxiceffects to the host cell, tissue, and the patient (in the case of invivo use).

Intracellular delivery and transfection in accordance with the methodsof the present invention may be performed in vivo, ex vivo, and invitro. Included within the above three methods is human gene therapyincluding wherein cells to be treated are excised from a patient. Thecells are treated with an appropriate nucleotide sequence andtransfection with ultrasound is carried out in cell culture. Thetransfected cells are analyzed for gene expression of the appropriateprotein. The successfully transfected cells, measured by geneexpression, are then returned to the body of the patient. Transfectionwith ultrasound thereby results in the treatment of diseases by genetherapy. Diseases to be treated with the methods of the presentinvention include and are not limited to acquired immune deficiencysyndrome, autoimmune diseases, chronic viral infection, hemophilia,muscular dystrophy, cystic fibrosis, diabetes, atherosclerosis, livercancer, lung cancer, prostate cancer, ovarian cancer, brain cancer,kidney cancer, melanoma, neuroblastoma, and breast cancer. Many otherdiseases may, of course, be treated with the methods of the presentinvention, as will be apparent to the skilled artisan upon reading thepresent disclosure, and the treatment of all such diseases are to beconsidered within the scope of the present methods.

The use of heat, for example in the form of ultrasound, lithotripsyshock waves, and increased body temperature, in the present invention isuseful in aiding the delivery of compounds, such as, for example,nucleotide sequences, into cells for therapeutic purposes. Theintroduction of a nucleotide sequence into the cell is the first step inincorporating the sequence into the genome. Such transfection techniquesmay be useful in conjunction with testing the range of ultrasoundfrequency useful in inducing the delivery of compounds, includingnucleotide sequences, to cells.

Each of the methods of the present invention include administering allor part of a sense or an antisense sequence for insulin (Giddings andCarnaghi, Mol. Endocrinol. 1990 4:1363-1369), Bcl 2 (Tsujimoto, Y., etal., PNAS, USA 1986, 83:5214-5218), human leukocyte antigen (Trucco, G.,et al., Diabetes 1989, 38:1617-1622, thymidine kinase (Axel, R., et al.,J. Supramol. Struct. 1979, 8 (Suppl. 3):41), HLA-B7, Factor VIII(Higuchi, M., et al., Genomics 1990, 6:65-71, ras/p53 (Arai, N., et al.,Mol Cell Biol 1986, 6:3232-3239, Mitsudomi, T., et al., Chest 1993,104:362-365), high density lipoprotein (hdl), leutinizing hormonereleasing hormone (Maier, C. C., et al., Cell Mol Neurobiol 1992,12:447454) and leutinizing hormone releasing hormone antagonist,antitumoral agents such as and not limited to insulin-like growthfactor-1 (IGF-1, Barnes, M., et al., Obstetrics and Gynecology 1997,89:145-155), anti-IGF-1 (human IGF-1 gene fragment from published patentapplication GB2241703 GenBank accession number A29119), anti-k-ras (dogspleen mRNA 212 nucleotides GenBank accession number S42999), anti-c-fos(Rattus norvegicus Sprague Dawley c-fos gene, 5′ flanking region GenBankaccession number U02631), bcr-abl (Barnes, M., et al., Obstetrics andGynecology 1997, 89:145-155), c-myc (mouse c-myc gene, exons 1 and 2GenBank accession number L00038, J00373, and J00374), c-myc promoter(Barnes, M., et al., Obstetrics and Gynecology 1997, 89:145-155), erbB-2promoter (Barnes, M., et al., Obstetrics and Gynecology 1997,89:145-155), erbB2 promoter-cytosine deaminase (human c-erb B2/neuprotein gene, partial cDNA (cds) GenBank accession number M95667), andantivirals such as and not limited to anti-human papilloma virus (HPV),anti-human immunodeficiency virus (HIV) such as HIVenv+rev (HIV type 1,isolate BTSPR, env gene, C2V3 region, partial cds GenBank accessionnumber U53195), tar/Td-rev (HIV type 1 rev-1 gene, 5′ end GenBankaccession number M38031, synthetic HIV1 TAR, 5′ end GenBank accessionnumber M27943), ribozyme, zeta-chimpanzee receptor, and the like, andall or part of a sequence encoding cytokines such as and not limited tointerleukin 2 (IL-2) (human brain MRNA 418 nucleotides GenBank accessionnumber S77835), interleukin 4 (Arai, N., et al., J Immunol 1989,142:274-282), interleukin 7 (human gene, exon 1 GenBank accession numberM29048), interleukin 12 (mouse 5′ flanking region of IL-12 p35 geneGenBank accession number D63334), interleukin 4 (human IL-4 gene,complete cds GenBank accession number M23442), interleukin 6 (human genefor nuclear factor NF-IL-6 GenBank accession number X52560); gp130 (LIFreceptor/IL-6 receptor complex component MRNA 150 nucleotides GenBankaccession number S80479), interleukin 6 receptor, granulocyte macrophagecolony stimulating factor (GM-CSF) (human GM-CSF gene, 5′flanking/promoter region GenBank accession number U31279), interferonincluding interferon gamma (human immune IFN-γ gene, complete cdsGenBank accession number J00219, M37265, V00536), tumor necrosis factorbeta, TNF-β, (human 5′ sequence of TNF-α gene GenBank accession numberX59351)), vascular endothelial growth factor (VEGF), human growthhormone (hGH, Fidders, J. C., et al., Proc Natl Acad Sci (USA) 197976:4294-4298), colony stimulating factor, Factor VIII, Factor IX, FactorX, and the like. Other sequences useful in the methods of the presentinvention include ribozymes including catalytic RNA which may have ahammerhead secondary structure (Bratty, et al., Biochim. Biophys. Acta1993 1216:345-349 and McKay, D. B., RNA 1996 2:395-403), c-myc, c-myb,tumor suppressor genes such as and not limited to human tumor antigenp53 (5′ end GenBank accession number M26864), genes offeringchemoprotection such as and not limited to those encoding multidrugresistance protein (MDR) (human mdr1 gene GenBank accession numberX78081), genes for antigen overexpression such as and not limited toHLA-B7 (beta 2 microglobulin) (mouse MHC class I HLA-B7 gene, 5′flanking region GenBank accession M35971), carcinoembryonic antigen(CEA) (human 5′ region GenBank accession number U17131), suicide genessuch as and not limited to thymidine kinase (TK) (human TK gene encodingTK and promoter region GenBank accession number M13643), Ras, genecomplementation genes such as and not limited to cystic fibrosistransmembrane conductance regulator (CFTR) (human CFTR gene, exon 1GenBank accession number M55106 and M55499), adenosine deaminase (ADA)(human ADA gene, complete cds GenBank accession number M13792),glucocerebrosidase, IRAP/TK (human MRNA for IRAP GenBank accessionnumber X53296), vascular endothelial growth factor (VEGF) (mus musculusVEGF gene, partial cds and promoter region GenBank accession numberU41383), LDLR (human LDL receptor gene fragment GenBank accession numberM60949), Fanconi Anemia Complementation Group C (FACC) (human FACC gene,5′ region GenBank accession number X83116), p47-phox (human P47 LBConcogene MRNA, complete cds GenBank accession number U03634), Factor IX(human Factor 1× gene, exon 1 GenBank accession number K02048), α-1antitrypsin (human α-1 antitrypsin gene S variant, complete cds GenBankaccession number K02212), α-1 iduronidase (human iduronidase genesequence GenBank accession number M88001), and iduronate sulfatase (Ids)(Mus musculus Ids MRNA, complete cds GenBank accession number L07921),and gene markers such as and not limited to NeoR and LacZ,(bacteriophage T4 td gene, exon 2, 3′ end; ORF2, complete cds and ORF3,5′ end GenBank accession number M22627, and cloning vector pZEO (isolateSVLacZ) β-galactosidase (lacZ) gene, phleomycin/zeocin-binding protein(ShBle) gene, (complete cds GenBank accession number L36850).

DNA encoding certain proteins may be used in the treatment of manydifferent types of diseases. For example, adenosine deaminase may beprovided to treat ADA deficiency; tumor necrosis factor and/orinterleukin-2 may be provided to treat advanced cancers; HDL receptormay be provided to treat liver disease; thymidine kinase may be providedto treat ovarian cancer, brain tumors, or HIV infection; HLA-B7 may beprovided to treat malignant melanoma; interleukin-2 may be provided totreat neuroblastoma, malignant melanoma, or kidney cancer; interleukin-4may be provided to treat cancer; HIV env may be provided to treat HIVinfection; antisense ras/p53 may be provided to treat lung cancer; andFactor VIII may be provided to treat Hemophilia B. See, for example,Thompson, L., Science, 1992, 258, 744-746. Nucleotide sequences for theabove-identified proteins are available in the scientific literature,including GENBANK, and are known to skilled artisans.

In addition to a coding sequence or antisense sequence, the nucleotidesequence administered to cells may have additional sequences to assistin the expression of the sequence. Suitable expression vectors,promoters, enhancers, and other expression control elements are known inthe art and may be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989). Promoters such as and not limited toSV40, RSV, CMV, cd5k, IL5Rα pgk-1, srα, TK, and the like are useful inthe present invention. Transcription and/or translation control elementsmay be operatively linked to the sequence. For example, in an upstreamposition, a promoter may be followed by a translation initiation signal,comprising a ribosome binding site and an initiation codon, and in adownstream position may be a transcription termination signal. Thetranscription and translation control elements may be ligated in anyfunctional combination or order. The transcription and translationcontrol elements used in any particular embodiment of the invention willbe chosen with reference to the type of cell into which the expressionvector will be introduced, so that an expression system is created. Theselection of promoters, enhancers, and other expression control elementsand the preparation of expression vectors suitable for use in thepresent invention will be well within the ambit of one skilled in theart once armed with the present disclosure. Also, introduction of theexpression vector incorporating a sequence into a host cell can beperformed in a variety of ways known in the art.

Mammalian cells may be primed to be more susceptible to uptake of DNAfor gene therapy by the addition of various media, buffers, andchemicals known to those of skill in the art and set forth in Sambrook,supra. Administration of nucleotide sequences in vivo may include, ifdesired, more than one sequence. For example, a single carrier maycontain more than one sequence or carriers containing differentsequences may be co-administered. In addition, one sequence may bedelivered in a carrier and another naked sequence coadministered.Additional sequences, such as promoter sequences, may be deliveredtogether with a sequence for therapeutic delivery, to increaseexpression thereof. For example, a heat shock protein nucleic acidsequence is an example of an upregulating gene sequence which may beused to increase expression of a second gene sequence.

A wide variety of compounds (in addition to genetic material) may alsobe delivered to cells in accordance with the methods of the invention.Such other compounds include various other bioactive agents. As usedherein, “bioactive agent” refers to any substance which may be used inconnection with an application that is therapeutic or diagnostic innature, such as, for example, in methods for diagnosing the presence orabsence of a disease in a patient or in methods for the treatment ofdisease in a patient. As used herein, “bioactive agent” refers also tosubstances which are capable of exerting a biological effect in vitro,in vivo, and/or ex vivo. The bioactive agents may be neutral, orpositively or negatively charged, etc., as desired. Examples of suitablebioactive agents include diagnostic and pharmaceutical agents, includingdrugs, synthetic organic molecules, proteins, peptides, vitamins,steroids, steroid analogs; and also include genetic material, includingnucleosides, nucleotides and polynucleotides.

The phrase “diagnostic agent”, as used herein, refers to any agent whichmay be used in connection with methods for imaging an internal region ofa patient and/or diagnosing the presence or absence of a disease in apatient. Exemplary diagnostic agents include, for example, contrastagents for use in connection with ultrasound imaging, magnetic resonanceimaging or computed tomography imaging of a patient. Diagnostic agentsmay also include any other agents useful in facilitating diagnosis of adisease or other condition in a patient, whether or not imagingmethodology is employed.

The terms “pharmaceutical agent” or “drug”, as employed herein, refer toany therapeutic or prophylactic agent which may be used in the treatment(including the prevention, diagnosis, alleviation, or cure) of a malady,affliction, disease or injury in a patient. Therapeutically usefulpeptides, polypeptides and polynucleotides may be included within themeaning of the term pharmaceutical or drug, as are various othertherapeutically useful organic or inorganic compounds.

Particular examples of pharmaceutical agents which may be delivered bythe methods of the present invention include, but are not limited to:mitotic inhibitors such as the vinca alkaloids, radiopharmaceuticalssuch as radioactive iodine, phosphorus and cobalt isotopes; hormonessuch as progestins, estrogens and antiestrogens; anti-helminthics,antimalarials and antituberculosis drugs; biologicals such as immunesera, antitoxins and antivenins; rabies prophylaxis products; bacterialvaccines; viral vaccines; aminoglycosides; respiratory products such asxanthine derivatives, theophylline and aminophylline; thyroidtherapeutics such as iodine salts and anti-thyroid agents;cardiovascular products including chelating agents and mercurialdiuretics and cardiac glycosides; glucagon; blood products such asparenteral iron, hemin, hematoporphyrins and their derivatives;targeting ligands such as peptides, antibodies, and antibody fragments;biological response modifiers such as muramyl dipeptide, muramyltripeptide, microbial cell wall components, lymphokines (e.g. bacterialendotoxin such as lipopolysaccharide and macrophage activation factor);subunits of bacteria (such as Mycobacteria and Cornebacteria); thesynthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine; antifingalagents such as ketoconazole, nystatin, griseofulvin, flucytosine (5-fc),miconazole, and amphotericin B; toxins such as ricin; immunosuppressantssuch as cyclosporins; and antibiotics such as β-lactam and sulfazecin;hormones such as growth hormone, melanocyte stimulating hormone,estradiol, beclomethasone dipropionate, betamethasone, betamethasoneacetate, betamethasone sodium phosphate, betamethasone disodiumphosphate, betamethasone sodium phosphate, cortisone acetate,dexamethasone, dexarnethasone acetate, dexamethasone sodium phosphate,flunisolide, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, paramethasone acetate,prednisolone, prednisolone acetate, prednisolone sodium phosphate,prednisolone tebutate, prednisone, triamcinolone, triamcinoloneacetonide, triamcinolone diacetate, triamcinolone hexacetonide,fludrocortisone acetate, oxytocin, and vasopressin, as well as theirderivatives; vitamins such as cyanocobalamin neionic acid; retinoids andderivatives such as retinol palmitate and α-tocopherol; peptides andenzymes such as manganese superoxide dismutase and alkaline phosphatase;anti-allergens such as amelexanox; anti-coagulation agents such asphenprocoumon and heparin; tissue plasminogen activators (TPA),streptokinase, and urokinase; circulatory drugs such as propranolol;metabolic potentiators such as glutathione; antibiotics such asp-aminosalicyclic acid, isoniazid, capreomycin sulfate cycloserine,ethambutol hydrochloride ethionamide, pyrazinamide, rifampin,streptomycin sulfate dapsone, chloramphenicol, neomycin, ceflacor,cefadroxil, cephalexin, cephadrine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxicillin, cyclacillin, picloxicillin, hetacillin, methicillin,nafcililn, oxacillin, penicillin (G and V), ticarcillin rifampin andtetracycline; antivirals such as acyclovir, DDI, Foscarnet, zidovudine,ribavirin and vidarabine monohydrate; antianginals such as diliazem,nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate,nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate;antiinflammatories such as difluisal, ibuprofin, indomethacin,meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin, and salicylates;antiprotozoans such as chloraquine, hydroxychloraquine, metranidazole,quinine and meglumine antimonate; antirheumatics such as penicillamine;narcotics such as paregoric; opiates such as codeine, heroin, methadone,morphine, and opium; cardiac glycosides such as deslanoside, digitoxin,digoxin, digitalin, and digitalis; neuromuscular blockers such asatracurium nesylate, gallamine triethiodide, hexaflorenium bromide,metrocurine iodide, pancurium bromide, succinylcholine chloride(suxamethonium chloride), tubocurarine chloride and vecuronium bromide;sedatives such as amorbarital, amobarbital sodium, aprobarbital,butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate,flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride,methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital,pentobarbital sodium, secobarbital sodium, tulbutal, temazepam andtrizolam; local anesthetics such as bupivacaine hydrochloride,chloroprocaine hydrochloride, etidocaine hydrochloride, lidocainehydrochloride, mepivacaine hydrochloride, procaine hydrochloride, andtetracaine hydrochloride; general anaesthetics such as droperidol,etamine hydrochloride, methohexital sodium and thiopental sodium;antineoplastic agents such as methotrexate, fluorouracil, adriamycin,mitomycin, ansamitomycin, bleomycin, cystein arabinoside, arabinosyladenine, mercaptopolylysine, vincristine, busulfan, chlorambucil,azidothymidine, melphalan (e.g. PAM, L-PAM or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin(actinomycin D), danorubicin hydrochloride, dosorubicin hydrochloride,taxol, plicamycin (mithramycin), aminoglutethimide, estramustinephosphate sodium, flutamide, leuprolide acetate, leuprolide acetate,megestrol acetate, tamoxifen citrate, testolactone, trilostane,amsacrine (m-AMSA), asparaginase, etoposide (VP-16), interferon α-2a,interferon α-2b, teniposide (VM-26), vinblastine sulfate (VLB),vincristine sulfate, hydroxyurea, procarbaxine, and dacarbazine.

Although a wide variety of compounds, including nucleotides, may bedelivered in accordance with the present invention, preferably thenucleotides are less than about 10,000 bases (or base pairs) in length,more preferably between about 20 to about 10,000 bases (or base pairs)in length, even more preferably between about 2,000 and about 8,000bases (or base pairs) in length, and most preferably between about 4,000and 6,000 bases (or base pairs) in length. Other (non-nucleotide)compounds or bioactive agents to be delivered are preferably less thanabout 5000 kilodaltons (5000 kD) in molecular weight, more preferablybetween about 10 and about 1000 kD, even more preferably between about100 and about 500 kD. As one skilled in the art will recognize, however,larger and smaller sized compounds may also be delivered in accordancewith the present invention.

The useful dosage of nucleotide sequences or other compounds to beadministered or delivered, as well as the mode of administration, willvary depending upon type and nature of the compound to be delivered, theage, weight, cells or patient (animal) to be treated, the particulardiagnostic, therapeutic, or other application intended (including thedisease state, if any, to be treated), and the organic halide (if any)and carrier (if any) employed. Typically, dosage is initiated at lowerlevels and may be increased until the desired therapeutic effect isachieved. The desired dosage, including any therapeutically ordiagnostically effective dosage amounts, will be well within the ambitof one skilled in the art, armed with the prevailing medical literatureand with the present disclosure. Representative amounts are provided inthe examples herein. Of course, higher or lower amounts may be employed,as will be recognized by the skilled artisan.

As one skilled in the art would recognize, administration ofcompositions of the present invention may be carried out in variousfashions, such as intravascularly, intralymphatically, parenterally,subcutaneously, intramuscularly, intranasally, intrarectally,intraperitoneally, interstitially, into the airways via nebulizer,hyperbarically, orally, topically, or intratumorly, using a variety ofdosage forms. One method of topical administration is the addition of anucleotide sequence (or other compound to be delivered), preferably in acarrier such as and not limited to a hydrogel, applied to the outside ofa balloon catheter. The catheter is inserted into the blood stream of apatient. Once the balloon of the catheter reaches the location to whichthe sequence is to be administered, the balloon is pumped up and thesequence-containing hydrogel adheres to the blood vessel surface thusdelivering the sequence. In addition, ultrasound may be applied to thecells endoscopically and intravascularly, for example, as well as, ofcourse, applied externally.

A number of transfection and other intracellular delivery techniques arepossible in accordance with the methods of the present inventionemploying the subject methods and the organic halides and/or carriers asdisclosed herein. Two methods, using calcium phosphate and viralvectors, are indirect methods of introducing the nucleotide sequenceinto cells because they involve the passive uptake of the nucleotidesequence by the cell which is to be transfected.

Calcium phosphate coprecipitation is a chemical-mediated indirect methodof transfection. The nucleotide sequence (or other compound to beadministered) is introduced into mammalian cells, for example, bycoprecipitation of the sequence with calcium phosphate, calciumchloride, calcium hydroxybutarate, and the like; then the mixture ispresented to the cells. The purified nucleotide sequence is mixed withbuffers containing phosphate and calcium chloride which results in theformation of a very fine precipitate, and the mixture is presented tothe cells in culture. A protocol for cells that grow attached to asubstratum as set forth in Keown, W. A., et al., “Methods forIntroducing DNA into Mammalian Cells,” in Methods in Enzymology, Vol.185, Gene Expression Technology, Ed., Goeddel, David V., pp. 527-537,Academic Press, Inc., New York, N.Y., 1991 is incorporated herein byreference in its entirety. Briefly, on day 1, cells are seeded at2-3×10⁴ cells/cm² in normal growth medium and allowed to attach. At thetime of transfecting, the cells should be 80-90% confluent. On day 2,the nucleotide sequence-calcium phosphate copreciptate is prepared,mixed and allowed to stand at room temperature for about 30 minutes. Thenucleotide sequence is added to TE buffer (10 mM tris, 1 mM EDTA Ph8.0), 2×HBAS (Hanks' balanced slats, 1.4 mM Na₂HPO₄, 10 mM KCl, 12 mMglucose, 275 mM NaCl, and 40 mM HEPES, ph 6.95), and 2M CaCl₂ (calciumchloride in 10 mM HEPES, pH 5.8). The medium is removed from the cellsand replaced with fresh medium. The precipitate is mixed gently byshaking or pipetting and added directly to the medium in dishescontaining cells. The cells are incubated at 37° C. for 4 hours. Themedium containing the precipitate is removed and dimethyl sulfoxide in1×HBS. After 2 minutes, 4 ml of serum-free medium is added to each dish.The mixture is aspirated, washed twice with serum-free medium, andmedium is added and incubated overnight at 37° C. The cells aretrypsinized and the contents of each plate is split into 3-4 new plates.Selection may be applied for stable transfectants, in which selectivemedium may be used at this time or a day later.

The present invention employing the methods of the invention and theorganic halides and/or carriers may also be useful concurrently withmicroinjection and electroporation. Microinjection involves the directmicroinjection of nucleotide sequences into the nucleus of a host cell.Microinjection does not expose the nucleotide sequence to the cytoplasmor organelles within it. This is beneficial since considerable damagemay result to the DNA during transit from the cell exterior to thenucleus. Electroporation involves electric field-mediated nucleotidesequence transfection. When membranes are subjected to an electric fieldof sufficiently high voltage, regions of the membrane undergo areversible breakdown, resulting in the formation of pores large enoughto permit the passage of nucleotide sequences. Electroporated nucleotidesequences remain free in the cytosol and nucleoplasm. Very few copies oftransfected nucleotide sequences may be introduced with electroporation.Cells susceptible to electroporation include, for example, lymphocytes,hematopoietic stem cells, and rat hepatoma cells.

“Ultrasound”, “Sonoporation™”, and similar terms, refer to pulses ofsound energy, preferably repetitive pulses, sufficient to assist ininducing the delivery of a compound into a cell, and, if desired, theformation of a gas from a gaseous precursor. Preferably, the ultrasoundis in the frequency range of from about 10 kilohertz to less than about50 megahertz and at an energy level of from about 200 milliwatts/cm² toabout 10 watts/cm². While not intending to be bound to any particulartheory of operation, the ultrasound may assist in the delivery of thecompounds to the cells by inducing openings in the cell membrane, orperhaps bursting endosomes inside a cell allowing compounds to escape.Indeed, cells may be induced to take up (e.g., be transfected with)compounds (e.g., nucleotide sequences) with ease compared toconventional methods. Typically the ultrasound is applied by externalapplication, via a standard clinical ultrasound device, but may also beapplied in other fashions, such as endoscopically and intravascularly,as described above. The use of ultrasound in connection with the presentinvention may, in certain embodiments, be preferred. However, as notedherein, the use of ultrasound is not necessary or critical to theoperation of the methods of the invention. Thus, the subject methods maybe carried out with the application of ultrasound, or without theapplication of ultrasound, as desired.

In accordance with the present invention, for in vivo applications, alower frequency of sound is usually selected for cells of deep seated orthick tissues, e.g. transcutaneous application of ultrasound to cells ofthe deep seated muscle or organs in the abdomen or retroperitoneum. Forcells of small tissues a higher frequency of sound energy is applied,e.g. for the eye. For intravascular applications, which may employintravascular catheters equipped with ultrasound transducers forendovascular gene therapy, higher frequencies may be employed such asover about 20 megahertz. For most applications however the frequency ofthe sound ranges from about 500 kilohertz to about 3 megahertz,preferably from about 500 kilohertz to about 1 megahertz, morepreferably about 200 kilohertz, more preferably about 40 kilohertz toabout 25 megahertz, even more preferably about 10 megahertz. Compared tolithotripsy, the frequency employed in the present invention is morethan about 2 or 3 orders of magnitude higher and the energy levels ofthe present invention are lower.

The sound energy is applied in waves of sonic energy over a given dutycycle (sometimes referred to as pulse duration) and level of intensity.Generally continuous wave ultrasound which applies a constant train ofultrasound pulses is employed. The duty cycle is selected so that thelevel of energy output is in a desired range. The duty cycle may bevaried from between 1% and 100% meaning that the ultrasound energy willbe pulsing from between 1% and 100% of the time. For example, a periodof ultrasound treatment may take place over 25 minutes with three dutycycles of ultrasound, each five minutes in duration, interrupted by twoperiods of no ultrasound. Preferably the duty cycle is 100%, morepreferably about 75%, more preferably about 50%, even more preferablyabout 20%, even more preferably about 15%, and even more preferablyabout 10%.

Ultrasound for use in the present invention is typically provided at afrequency lower than the frequency used for imaging by ultrasound. Thefrequency of ultrasound which is selected will vary depending upon thelocation of cells which are being transfected, and or other factors thatwill be readily apparent to one skilled in the art based upon thepresent disclosure.

In addition to frequency, the energy level (sometimes referred to aspower intensity or power level) also has a large effect on total energywhich is applied to the cells or tissue for ultrasound enhancedtransfection. Suitable energy levels will be readily apparent to oneskilled in the art based upon the present disclosure. Typically, theenergy level settings are somewhat higher than employed in diagnosticultrasound but may range from about 500 milliwatts/cm² to about 10watts/cm², more preferably from about 200 milliwatts/cm² to about 10milliwatts/cm², and more preferably of from about 50 milliwatts/cm² toabout 2 watts/cm². The power level which is applied is selected so thatboth peak spatial temporal power and total energy deposition isgenerally below the cytotoxic threshold for the cells or tissue.Generally, frequencies and energy levels are applied at lower amounts,then increased until the desired cellular uptake of the administeredcompound is achieved.

As one skilled in the art would recognize, high energies of ultrasoundmay be used for hyperthermia to heat the tissue and also to directlyablate tissues with very high levels of energy. In the ultrasoundenhanced transfection and gene expression of the present invention,energy levels are far below those which cause tissue ablation and belowthose which cause a significant hyperthermic effect. As one skilled inthe art would recognize once armed with the present disclosure, energydeposition is a function of both power intensity and duty cycle. Higherspatial peak temporal average power tends to shift the bioeffect curvesuch that lower total energy may be applied to create a greaterbioeffect. Higher energy levels and lower ultrasonic frequencies arerequired for penetration into deep seated tissues; conversely lowerenergy levels and higher ultrasonic frequencies are needed for treatmentof superficial tissues or when the ultrasound transducer can be applieddirectly to the tissue surface. Small volume cell culture samples needless power for ultrasound enhanced transfection than large volumebioreactor chambers which may be multiple liters in size and thereforeneed higher energy levels to enhance gene expression. The geometry of acell culture container will also affect the ultrasound energyrequirements.

In accordance with the present invention, ultrasound energy may be usedto increase the efficiency of cellular uptake of a compound (e.g.transfection) by inducing a cell to take up a compound. In addition,gene expression of a nucleotide sequence is enhanced by the applicationof ultrasound.

The ultrasound energy may be applied to the tissue or cells eitherbefore, simultaneously with, or after administration of the compound tothe cell, preferably simultaneously with or after. Typically theultrasound energy is applied from no more than about 48 hours prior toadministration of the compound or genetic material to the cells and/orup to no more than about 48 hours after the genetic material has beenadministered to the cell, although longer or shorter times may beapplied. More preferably, the ultrasound energy is applied at some timeor at various time points from about 4 hours before administration ofthe compound or genetic material up to about 24 hours afteradministration. Most preferably the ultrasound energy is applied withinabout 1 hour prior to transfection up to about 12 hours posttransfection.

Either one or multiple applications of ultrasound energy may beemployed. The duration of ultrasound energy exposure (exposure time)will vary depending upon the power level of the ultrasound and the dutycycle. To determine the preferable duration, ultrasound is typicallyapplied at lower exposure times, and increased until the desiredcellular uptake of the compound administered is achieved. A highintensity (high power level; typically greater than about 2 watts/cm²,preferably over about 5 watts/cm², and also preferably over about 10watts/cm², depending on the pulse duration) ultrasound shock wave mayrequire only a few milliseconds of exposure. This may also be the casewhen cavitation nuclei such as gas filled liposomes or perfluorocarbonemulsions are present within the medium. A very brief exposure to highenergy ultrasound may be sufficient to enhance transfection. Thepresence of cavitation nuclei in the transfection medium will lower thecavitation threshold and therefore potentially decrease energyrequirements for ultrasound enhanced transfection as well as topotentially decrease the necessary exposure time. More typically theexposure time ranges from about a few seconds to up to about an hour ofultrasound energy application to the cell to achieve most effectiveultrasound enhanced gene tmnsfection. Even more preferably the durationof ultrasound exposure ranges from about a few seconds to about a fewminutes and may be repeated at various intervals during transfection.The duration of ultrasound energy exposure should be sufficient to causethe desired effect but not so long that significant cytotoxicity mayresult.

The effect of ultrasound enhanced transfection is independent ofhyperthermia. While the application of ultrasound energy necessary toincrease the efficiency of transfection may result in a few degreescentigrade increase in temperature, any increase in temperature istypically transient and the temperature rapidly returns to baseline.More preferably the temperature does not increase significantly duringapplication of the ultrasound. An increase in temperature is typicallyless than about 1° C. to about 2° C. Progressively higher levels ofultrasound energy will result in progressive rises in temperature buttemperature is preferably maintained below the level where significantcytotoxicity will occur (e.g. 44° C. or higher). As one may note, thesample measures the temperature in a solution of normal saline whenexposed to ultrasound. The applied energy is 10 watts imparted through a5.0 cm² transducer, or 2 W cm⁻². Sound energy from the ultrasoundtransducer may be simply converted to thermal energy in the aqueousmilieu. The amount of energy and/or the time of exposure may be modifiedso as to prevent temperature-induced cell destruction.

The ultrasound energy may be applied with any of a variety ofcommercially available ultrasound systems. For example a Rich-Mar model25 ultrasonic therapy apparatus (Rich-Mar Corporation, Inola, Okla.)with the center frequency residing at approximately 1.0 Mhz, in pulsedor continuous mode, may be used to practice the invention.Conventionally available transducers, power amplifiers and othercomponent systems for practicing the invention can also be readilyassembled. Wave synthesizers and pulsers may also be incorporated intothe system to allow control over the pulse repetition intervals, dutycycles, etc. Advantageously, these components can also be used to modifythe ultrasound pulses to employ varying frequency and amplitude effectssuch as CHIRP (increasing in frequency) and PRICH pulses (decreasing infrequency) waveform patterns. Ultrasonic energies can also be suppliedfrom commercially available amplifiers, transducers and frequencygenerators. By way of example, a power transducer with a centerfrequency of 1.0 Mhz from Valpey-Fisher (Valpey-Fisher, Hopkinton,Mass.), a power RF amplifier from ENI (ENI, Rochester, N.Y.), and afunction generator from Hewlett Packard (Hewlett Packard, Sunnyvale,Calif.) may be a suitable setup to accomplish the above goals.Alternatively, a pulse/function generator or an arbitrary functiongenerator may also be used to accomplish variable pulse formats. Inaddition, methods that would allow for gating the various signalstogether, could conceivably be accomplished.

The high energy ultrasound system may also be incorporated withultrasound imaging such as described in U.S. patent application U.S.Ser. No. 08/468,052, filed Jun. 6, 1995, and the disclosures of whichare hereby incorporated herein by reference in their entirety. Alsoapplication of high energy ultrasound may be performed under other formsof conventional imaging such as endoscopy (e.g. fiberoptic), computedtomography, magnetic resonance imaging, angiography, and nuclearmedicine. Such imaging may be employed, for example, to locate andidentify in a patient the cells to which the ultrasound induced (orother) heating should be applied, or used to follow and/or locate thecomposition of the invention after administration to a patient.

The ultrasound may be applied so as to effectively create secondharmonic superimposition on the target treatment zone of tissue toincrease the effectiveness of transfection. For example, a prototypesector-vortex phased array transducer, depicted in FIG. 5, 120 mm indiameter, which generates 750 kHz and 1.5 MHz ultrasound may beemployed. As described in the reference by K. Kawabata and S. Umemura,Ultrasonics Sonochemistry 1996, 3:1-5, a transducer may be constructedwith 32 piezoelectric (PZT) transducer elements from lead zirconate PZTmaterial. The transducer may be constructed in two tracks such thatthere are 16 sectors in each track. The lower frequency ultrasound couldbe applied from the outer track and the higher frequency, 1.5 MHz, fromthe inner track. A shell may be constructed with a 120 mm radius ofcurvature for geometric focusing. The beam profile provided by thepiezoelectric elements and spherical shape of the transducer cellassembly can be designed so as to superimpose the focal zones of the twodifferent frequencies of ultrasound. This may result in focal acousticpower with superimposition of the lower frequency and higher frequencyultrasound sources. This results effectively in second harmonicsuperimposition of the ultrasound signal. While not intending to bebound by any particular theory of operation, it is believed that thisultrasound assembly will allow for improved transfection efficiency atlower total amounts of energy and thus result in reducing damage to thecell.

Skilled artisans would recognize, once armed with the presentdisclosure, that the two ultrasound energy sources may be at otherfrequencies such that the first source (low frequency) is one half thefrequency of the second source. For example, 500 kHz in the outerassembly and 1 MHz in the inner assembly may be employed. A range ofdifferent frequencies may be selected such that the outer assembly is 1×and the inner assembly is 2×. Alternatively, the assemblies may bedesigned such that the higher frequency is in the outer assembly and thelower frequency is in the inner assembly. Alternatively still, oddharmonics may be utilized such that the outer and inner tracks may berepresented by X and 3× frequencies or X and 5× frequencies. The secondharmonic, ultraharmonic, or subharmonic frequencies are superimposed atthe focal zone which is directed towards the target tissues or cells tobe transfected. Thus, ultrasound may be administered simultaneously attwo or more frequencies to result in superimposition of ultrasonicfrequencies, including and not limited to second harmonic frequencies.

The present invention is also directed to a pharmaceutical kit whichcomprises a compound to be delivered, an organic halide and/or a carrier(including combinations thereof) for use to those desirous of deliveringto a cell a compound. The compound, organic halide, and/or carrier maybe mixed together or separately provided (as in, for example, separatecontainers, such as separate vials or packets). The pharmaceutical kitmay further comprise conventional kit components known to those skilledin the art once armed with the present disclosure, such as, mixingvials, syringes, gauze, etc.

The invention is further demonstrated in the following actual Examples1-3, 10-19, and 23, and prophetic Examples 4-9, and 20-22. The examples,however, are not intended to in any way limit the scope of the presentinvention.

EXAMPLES Example 1 Effect of Ultrasound on the Temperature of anAqueous-Based Medium in Culture Plate Well Phantoms and on CellViability

The first phase of evaluating the effect of ultrasound was to measurethe amount of heating caused by the ultrasound energy. The experimentalprotocol was designed to evaluate the heating in an individual well of a6 well culture plate while exposed to ultrasound. Ultrasound was appliedfor 30 seconds to each well and the relationship between energy andheating is shown in Table 2 below. TABLE 2 Temperature increaseTemperature increase Energy at 10% Duty Cycle at 100% Duty Cycle 0.5W/cm²   0° C. 0.5° C. 1 W/cm² 0° C. 1.5° C. 2 W/cm² 0.5° C.   2.9° C.

A follow-up experiment was carried out to assay the cell viability afterultrasound exposure. A cell proliferation kit using sodium3′-[1-(phenylamino-carbonyl)3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT, Boehringer-Mannheim, Indianapolis, Ind.) asthe cell viability indicator was carried out to evaluate whether celldamage had occurred. In this experiment, higher absorbance is due toviable cells causing uptake of XTT. Table 3 contains the results fromthis study. TABLE 3 Cell Viability as a Function of Ultrasound PowerInput Treatment Mean absorbance Standard Deviation No Ultrasound 7.830.41 0.5 W/cm² 10% Duty Cycle 7.83 0.41 0.5 W/cm² 100% Duty Cycle 7.830.41 1 W/cm² 10% Duty Cycle 7.17 0.75 1 W/cm² 100% Duty Cycle 6.00 1.411.5 W/cm² 10% Duty Cycle 7.67 0.82 1.5 W/cm² 100% Duty Cycle 1.83 0.98 2W/cm² 10% Duty Cycle 6.83 0.75 2 W/cm² 100% Duty Cycle 1.60 0.55

As the data in Table 3 shows, the first noticeable change in cellviability occurs with an energy of 1 W/cm² and a 100% Duty Cycle. Athigher energies with a 100% duty cycle a significantly larger number ofcells are destroyed.

Example 2 Effect of Ultrasound on Gene Expression in Cell Culture

Materials and Methods for Transfection and Measurement of GeneExpression

The DNA plasmid used was pCAT Control (GenBank accession number X65321)(Promega, Madison, Wis.) (see FIG. 3).

The plasmid was transformed into DH5-α Escherichia coli competent cells(Life Technologies, Gaithersburg, Md.). The cells were plated on LB agarplates (Bio 101, Vista, Calif.) that contained ampicillin (BoehringerMannheim Biochemicals (BMB), Indianapolis, Ind.). Resistant colonieswere selected, grown up and a Wizard mini-prep (Promega) plasmid DNAextraction was carried out. Plasmid DNA was cut with restriction enzymeEcoRI (BMB) and run on a 1% agarose gel (BMB). After fragments wereevaluated for size, the remaining culture was used to start a largeculture and a Wizard maxi-prep was carried out to produce DNA fortransfection. The DNA was quantified using a Hoefer TKO-100 mini-DNAfluorometer. The DNA was then ready for use in transfections.

Cationic liposomes were made by mixing dipalmitoyl ethylphosphocholineand dioleolyl phosphoethanolamine (Avanti Polar Lipids Alabaster, Ala.).The lipid mixture was resuspended in water and then sonicated to formsmall liposomes.

A human cervical cancer cell line (HeLa) was obtained from the AmericanType Culture Collection (Rockville, Md.) and grown in EMEM culture media(Mediatech, Washington, D.C.) supplemented with calf serum (LifeTechnologies). These cells were used in the transfections.

The DNA/lipid complex was formed in HEPES buffered saline (HEPES 20mmol/l, NaCl, 150 mmol/l, pH 7.4) (Sigma, St. Louis, Mo.) by mixing thelipid and DNA at a ratio of 6 parts lipid to one part DNA. This wasincubated for 30 minutes at room temperature and then used fortransfection.

The pCAT control plasmid encodes for the enzyme chloramphenicol acetyltransferase (CAT). This enzyme is not found in mammalian systems. TheCAT expression is assayed using a CAT ELISA kit (BMB). Thisnon-radioactive kit allows for sensitive detection of CAT expression.The kit is based on a sandwich of antibodies. A 96 well plate is coatedwith anti-CAT, this antibody binds the CAT in the sample. The nextantibody is the anti-CAT-digoxigenin, digoxigenin is a hapten found onlyin the digitalis plant. The rarity of this compound makes it ideal fornon-radioactive labelling. The next antibody added is ananti-digoxigenin that has been labelled with horseradish peroxidase.Horseradish peroxidase breaks down a substrate and causes a colorreaction which is then read with an SLT Spectra Shell plate reader(SLT-Labinstnrunents Ges.m.b.h., Groedin/Salzburg, Austria). Using astandard curve, this plate reader allows for measurement of proteinconcentration.

Protocol

The DNA complex was formed and added at a concentration of 30 μg oflipid and 5 μg of DNA per well in a 6 well plate containing HeLa cellsin 4 mls of EMEM. A Rich-Mar model 25 therapeutic ultrasound machine(Rich-Mar Corporation, Inola, Okla.) was used to apply ultrasound to thewells of the 6 well plate. The ultrasound was applied either 30 minutesbefore the DNA/lipid complex was added, 1 hour after the complex wasadded or 4 hours after the complex was added. In the first experiment, astandoff pad was used that covered the entire base of the 6 well plateand allowed sound to conduct from one well to another. The power settingwas 0.5 w/cm² with a 10% duty cycle.

Three conditions were tested. No ultrasound, ultrasound applied 30minutes before the transfection and ultrasound applied 1 hour after thetransfection.

The results set forth in Table 4 are from a transfection with a 1:1 mixof dipalmitoylethyphosphocholine and dioleoyl phosphoethanolamine(Avanti Polar Lipids, Alabaster, Ala.). The transfection was carried outin the presence of serum. In addition, a negative control was addedwhich included cells grown up not transfected with the 1:1 mix ofdipalmitoylethyphosphocholine and dioleoyl phosphoethanolamine andwithout ultrasound treatment. TABLE 4 Quantification of Gene Expressionin Cells Exposed to Ultrasound Treatment Cat expression (ng/ml) negativecontrol 0 no ultrasound 15.876 Ultrasound 20.529 30′ pretreatmentUltrasound 1 hour 43.794 post treatment

This experiment was repeated with a standoff pad designed to isolate thewells from each other, see FIG. 1 and FIG. 4. The base of a 6 well platewas cut away to allow transducer access and a second 6 well plate wasinverted above it. 2% agarose was poured onto this mold to form astandoff pad. The standoff pad was constructed to allow for open (deadair) spaces between the portions of the standoff pad that contacted the6 well plate, such that each well was raised above the standoff pad on avertical support with open spaces under each of the wells where thestandoff pad was cut away. The vertical supports were made of 2% agaroseand conducted the sound from the ultrasound transducer. The transducerwas placed below the standoff pad and sound was projected up through thepad into the each of the wells of the 6 well plate. The 6 well plate wasplaced upright on the standoff pad such that the cells on the bottom ofthe well were close to the transducer. In both experiments, theexpression of the CAT protein was measured by CAT ELISA after 48 hoursof incubation. Ultrasound was applied at 1 W/cm² and 100% duty cycle.The ultrasound was applied for 30 seconds on each well.

The same transfection reagent was used as in the first example. The testconditions were no ultrasound, 30 minutes before transfection, 1 hourafter transfection and 4 hours post transfection. Again the transfectionwas carried out in the presence of serum. In addition, a negativecontrol was added which included cells grown up not transfected withCAT/lipid complex and without ultrasound treatment. The results areshown in Table 5. TABLE 5 Treatment Cat expression (ng/ml) negativecontrol 0 no ultrasound 5.339 Ultrasound 5.339 30′ pretreatmentUltrasound 1 hour post 10.078 treatment Ultrasound 4 hours post 4.988treatment

Example 3 Application Using DNA with a Lipid Carrier and a Cavitator

The cells and the DNA/lipid complex were prepared as in Example 2. Sixwell plates were seeded with HeLa cells and filled with 16 ml of mediaas in Example 2. The lipid added was increased to 135 μg to allow forthe increase in volume and the DNA was also increased to 22.5 μg perwell. One hour after the complex was added, 100 μl of a liposomecomprised of the lipids dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidylethanolamine coupled to polyethylene glycol 5000(DPPE-PEG5000), and dipalmitoylphosphatidic acid (DPPA), in a ratio ofabout 82%:8%: 10% (mole %) was added to each well. The DPPE-PEG5000 wascomprised of DPPE and PEG5000 in a ratio of about 20%:80% (weight %).PEG5000 refers to PEG having an average molecular weight of about 5000.In addition, a negative control was added which included cells grown upnot transfected with CAT/lipid complex and without ultrasound treatment.

The six well plate was then covered with a sheet of parafilm to preventleakage, the lid was replaced and the plate was inverted. By invertingthe plate, the gas filled liposomes (cavitator) were allowed to float upto the cells, now on top of the standoff pad, plate construct. Theultrasound was applied from the bottom, however, in this case, the soundwas propagated through the media to the cells. After the ultrasoundexposure the plates were returned to an upright position and theparafilm removed. The plates were then incubated for 4 hours and the CATELISA performed the results of which are set forth in Table 6. TABLE 6Treatment Cat expression in ng/ml Negative control 0 No ultrasound 4.5840.5 w/cm²/35% duty cycle 9.634 0.5 w/cm²/100% duty cycle 19.910 1W/cm²/100% duty cycle 9.811

Example 4 Industrial Applications of Ultrasound Enhanced Transfection

A large scale bioreactor vessel containing a free suspension of cells isequipped with a flow through chamber housing an ultrasound transducer.Plasmid DNA containing the gene of interest is added to the cellsuspension with and without an organic halide. As the cells circulatethrough the chamber 500 kilohertz ultrasound is applied with a 10percent duty cycle at an energy level of 200 milliwatts per cm².Enhanced gene expression is attained, both with and without the organichalide. By varying the rate of flow of the cell suspension through theflow through chamber the proper ultrasound exposure time is attained foroptimal transfection efficiency.

Example 5 Ex Vivo Enhancement of Gene Expression in Human Cells

Plasmaphoresis is used to harvest the T cells of a patient withmetastatic malignant melanoma. The T cells are placed in tissue cultureand incubated with granulocyte macrophage colony stimulating factor(GMCSF) to increase multiplication of the lymphocytes. After sufficientcell densities have been achieved the gene for interleukin-2 (IL-2) isadded to the cells with a cationic liposomal vector, with and without anorganic halide. One hour later 1 megahertz ultrasound energy is appliedat a power level of 0.5 watts with a 10% duty cycle for a duration of 5minutes. Twenty-four hours later the cells are infused back into thepatient in an effort to treat the metastatic tumors. Promising resultsin the form of a perceptible decrease in tumor mass are observed, bothwith and without the organic halide. Additional testing also revealsenhanced IL-2 expression in the treated cells.

Example 6 Therapeutic Applications of Ultrasound Mediated Gene Delivery:Duchenne's Muscular Dystrophy

In a patient with Duchenne's Muscular Dystrophy plasmid DNA encoding thegene for dystrophin is injected at multiple sites into the muscles ofthe thighs and legs, with and without an organic halide. Ultrasound isthen applied to the thighs and legs using silicone gel as couplantbetween the transducer and the patient's skin. The frequency is 200kilohertz with a 10% duty cycle and a power level of 1 watt. Thetransducer remains for about 2 to 3 minutes over any one location on theskin. Enhanced expression for the gene for dystrophin is attainedresulting in increased muscle strength, both with and without theorganic halide.

Example 7 Therapeutic Applications of Ultrasound Mediated Gene Delivery:Atherosclerotic Heart Disease

A patient with atherosclerotic disease has marked narrowing of the leftanterior coronary artery. A balloon angioplasty catheter coated with ahydrogel material binding the gene for vascular endothelial growthfactor (VEGF), both with and without an organic halide, is placed at thesite of arterial stenosis and the balloon is inflated to a pressure of 9atmospheres. An endovascular ultrasound catheter is placed inside thevessel at the region where the angioplasty was performed and ultrasoundenergy is applied. The frequency is 20 megahertz at 1 watt per cm² with15% duty cycle for 3 minutes. Enhanced gene expression of VEGF isobserved with diminished propensity to restenosis at the angioplastysite, both with and without the organic halide.

Example 8 Therapeutic Applications of Ultrasound Mediated Gene Delivery

Similarly to Example 7 angioplasty is performed in a patient using aballoon catheter coated with the gene for VEGF, and with and without anorganic halide, but in this case the ultrasound energy is appliedtranscutaneously with a 1 megahertz focused transducer equipped withboth imaging and therapeutic elements. The therapeutic 1 megahertz soundis applied at an energy level of 500 milliwatts/cm² using a 20% dutycycle for a period of 5 minutes. The energy is focused upon theangioplasty site. Again enhanced VEGF gene expression is observed anddecreased propensity to restenosis, both with and without the organichalide.

Example 9 Therapeutic Applications of Ultrasound Mediated Gene Delivery

Colonoscopy is performed in a patient with genetic predisposition tocolon cancer. A region of epithelial metaplasia is identified in thedescending colon. An ultrasound transducer equipped with a semipermeablemembrane and drug storage reservoir containing the gene for Bcl 2 with aliposomal vector, both with and without an organic halide, is positionedover the area of epithelial metaplasia and ultrasound energy is appliedat a frequency of 500 kilohertz with an energy level of 500milliwatts/cm² and 10% duty cycle for a period of 3 to 5 minutes, todeliver the Bcl 2 gene to the cells in the region. On follow-upcolonoscopy 8 weeks later the epithelial metaplasia has decreasedsignificantly, particularly where an organic halide is employed in theadministration process. Further testing reveals enhanced Bcl 2expression in the region, both with and without the organic halideemployed in the delivery process.

Example 10 Transfection Efficiency of DPEPC/DOPE with and withoutOrganic Halides

Dipalmitoylethylphosphocholine (DPEPC) (Avanti Polar Lipids, Alabaster,Ala.) was mixed with dioleoylphosphatidylethanolamine (DOPE) (AvantiPolar Lipids, Alabaster, Ala.) at a 1:1 (w:w) ratio in 10 ml ofdeionized water at a lipid concentration of 1 mg per ml. 100 microlitersof n-perfluorohexane (PCR, Inc., Gainesville, Fla.) was added and mixedby shaking for 5 minutes on a Heavy Duty #6 Wig-L-Bug (Crescent Dental,Lyons, Ill.). The mixture was then extruded five times through two 0.8μm filters in a Lipex Biomembranes Extruder Device (Vancouver, BC,Canada). Particles without a fluorinated organic halide were sonicatedfor 10 minutes at room temperature in a bath sonicator. The mixture wasthen diluted in HEPES buffered saline at a ratio of 3011 of lipid mix to701 of HBS per well. pCMVCAT (Life Technologies, Inc., Gaithersburg,Md.) containing the gene for chloramphenicol acetyl transferase and thepromoter from human cytomegalovirus (CMV) was used to transfect HeLacells. pCMVCAT may be produced in accordance with the methods set forthin Foeeking and Hofstetter, Gene 1986 45:101-105, incorporated herein byreference in its entirety.

The pCAT® (Promega, Montgomeryville, Pa.) basic vector lacks eukaryoticpromoter and enhancer sequences. This allows maximum flexibility incloning any putative regulatory sequences into the convenient multiplecloning sites.

Expression of CAT activity in cells transfected with this vector isdependent on insertion of a functional promoter upstream from the CATgene. Enhancer elements can be inserted at the BamH I site downstreamfrom the CAT gene. The vector map sequence reference points are multiplecloning sites (Hind III-Xba I) 2242-2271, SV40 small T antigen region3064-3917, CAT gene start site 2315, CAT gene stop site 2974, andβ-lactamase (Amp′) coding region 209-1069.

Plasmid DNA was diluted to 5 μg per 100 μl in HBS. The DNA and lipidmixes were combined and incubated for 45 minutes at room temperature.HeLa cells is (ATCC certified cell line 2 (CCL 2)) were plated at aconcentration of 4×10⁵ per well in Eagle's MEM with non-essential aminoacids and Earle's BSS, 90%; fetal bovine serum, 10%. 200 μl of thelipid/DNA complex were added to each well and incubated 48-72 hours inthe presence of serum. The process was repeated using n-perfluorohexanein volumes of 50, 25, and 12.5 microliters. CAT expression was assayedusing a CAT ELISA from Boehringer Mannheim Biochemical (Indianapolis,Ind.). The results are shown in Table 7. TABLE 7 Effect of VariousAmount of Organic Halide on Chloramphenical Transacetylase Expression inVarious Cell Lines OH Organic Halide Volume CAT Standard Lipids (OH) μlCell Line Expression Deviation Lipofectin None HeLa 59.624 6.828Lipofectamine None HeLa 27.917 5.112 Lipofectin None C127 0 1.582Lipofectamine None C127 0 3.164 Lipofectin None COS1 104.424 117.883Lipofectamine None COS1 305.392 23.254 Lipofectin None NIH3t3 0 1.582Lipofectamine None NIH3t3 5.766 8.809 DPEPC/DOPE None HeLa 28.946 11.852DPEPC/DOPE None C127 24.493 21.491 DPEPC/DOPE None COS1 4930.403 262.278DPEPC/DOPE None NIH3t3 36.368 21.952 DPEPC/DOPE Perfluorohexane 12.5HeLa 3642.958 229.17 DPEPC/DOPE Perfluorohexane 25 HeLa 126.976 19.126DPEPC/DOPE Perfluorohexane 50 HeLa 33.454 10.94 DPEPC/DOPEPerfluorohexane 100 HeLa 28.69 2.61 DPEPC/DOPE Bromononafluorobutane12.5 HeLa 1795.29 1054.14 DPEPC/DOPE Bromononafluorobutane 25 HeLa2725.611 1004.542 DPEPC/DOPE Bromononafluorobutane 50 HeLa 2634.161456.709 DPEPC/DOPE Bromononafluorobutane 100 HeLa 2703.144 600.767DPEPC/DOPE Perfluorohexane 12.5 C127 167.911 88.724 DPEPC/DOPEPerfluorohexane 12.5 COS1 5348.326 93.809 DPEPC/DOPE Perfluorohexane12.5 NIH3t3 1968.405 316.894* DPEPC/DOPE isdipalmitoylethylphosphatidylcholine:dioleylphosphatidylethanolamine;CAT expression units are ng/ml (protein).

Example 11 Transfection Efficiency of DPEPC with Organic Halides

An example of transfection of DPEPC with perfluorohexane and1-bromononafluorobutane (BNFB, Fluoroseal, Houston, Tex.) was carriedout. Particles were prepared as set forth in Example 10 except that theBNFB sample was cooled before and after shaking on the Wig-L-Bug to keepthe BNFB in the liquid state. The results are shown in Table 7.

Example 12 Improved Transfection in Cell Lines Normally Resistant toTransfection with Perfluorohexane

COS-1 cells (ATCC cell repository line 1650 (CRL 1650)) were propagatedin Dulbecco's modified Eagle's medium, 90% and fetal bovine serum, 10%and plated at a concentration of 1×10⁵ per well. NIH/3T3 cells (ATCCcell repository line 1658 (CRL 1658)) were propagated in Dulbecco'smodified Eagle's medium with 4.5 g/liter glucose, 90% and calf serum,10% and plated at a concentration of 1×10⁵ per well. C127:LT cells (ATCCcell repository line 1804 (CRL 1804)) were propagated in Dulbecco'smodified Eagle's medium with 4.5 g/liter glucose, 90% and fetal bovineserum, 10% and plated at a concentration of 1×10⁵ per well. DPEPC:DOPEwas prepared with perfluorohexane at a volume of 12.5 microliters andwithout perfluorohexane as Example 10. The lipid/DNA mixtures wereincubated with the cells for 72 hours as described in Example 10.Lipofectin and Lipofectamine (Life Technologies, Gaithersburg, Md.),commercially available controls, were used according to themanufacturer's instructions. The results are shown in Table 7.

Example 13 Transfection Using Cationic Microspheres Filled withPerfluorobutane Gas

A lipid solution with 1 mg per ml of lipid composed of 1:1 (w:w) DPEPCwith dipalmitoylphosphatidylethanolamine (DPPE) was prepared and placedin a 2 ml vial with a head space of perfluorobutane gas. The sampleswere shaken for 1 minute of an ESPE CapMix at 4500 RPM resulting in gasfilled lipid coated microspheres. To each sample pCMVCAT was added at aDNA concentration of 1 μg per 6 μg lipid. The process was repeated using1:1 w:w DPEPC with DOPE. The gas filled microspheres prepared fromDPEPC/DPPE (gel state saturated lipids) formed stable gas filledmicrospheres binding the DNA. When transfection experiments wererepeated substantially as outlined in Example 11, however, there was noevidence of any appreciable gene expression. When gas filledmicrospheres were prepared from the DPEPC/DOPE lipids (DOPE is liquidcrystalline state at physiologically relevant temperature) the particlecount was lower than for the DPEPC/DPPE vesicles. When DNA was added tothe lipids, the vesicles fell apart, indicating how cationic lipidmicrospheres composed of liquid crystalline state lipid are unstable tobind DNA when the interior of the microsphere is filled with a gas PFC(perfluorobutane) as opposed to a liquid PFC (see above).

Example 14 Transfection with Organic Halides

The experiment conducted in Example 12 was repeated except thatfluorinated organic halides were used in eight samples and eight sampleswere subjected to ultrasound. Sixty minutes following the incubation ofthe DNA/lipid complex with the cells, ultrasound was applied byimmersing the head of a lMz transducer directly to the top of the cellculture well and ultrasound was applied for 5 to 30 seconds at a 10%duty cycle. Control groups were cells not exposed to ultrasound with andwithout transfection materials. Table 8 indicates that organic halidesincrease the efficiency of ultrasound such that lower levels of energyare more effective. 1-bromononafluorobutane (BNFB) together withultrasound results in about a 50% enhancement of expression; whereasabout a 30% enhancement of expression is evident with perfluorohexane(PFC6) and ultrasound. TABLE 8 US Expression Lipids OH (sec.) Actual SDDPEPC/DOPE BNFB 0 7007 2084 DPEPC/DOPE BNFB 5 10442 610 DPEPC/DOPE BNFB15 9910 656 DPEPC/DOPE BNFB 30 9381 140 none — 0 −137 23 DPEPC/DOPE — 01647 396 DPEPC/DOPE — 5 6444 846 DPEPC/DOPE — 30 8167 1206 Lipofectamine— 0 389 125 Lipofectin — 0 −118 14 DPEPC/DOPE PFC6 0 7290 2235DPEPC/DOPE PFC6 5 9477 1017 DPEPC/DOPE PFC6 15 8545 401 DPEPC/DOPE PFC630 9630 827

In all cases above the cell line is NIH/3T3. Units of DNA expression areng/ml protein. Ultrasound was applied at 0.5 W/cm², 10% duty cycle.OH-organic halide, US (sec.)—ultrasound in seconds,BNFB—(1-bromonanofluorobutane), PFC6—(perfluorohexane), SD—standarddeviation.

As can be seen above the lipid mixture of DPEPC/DOPE carrying the generesults in above background expression compared to Lipofectin orLipofectamine. This expression is enhanced with the addition of either afluorocarbon or by ultrasound. The best results were obtained withfluorocarbons and ultrasound in combination.

Example 15 Transfection with Perfluoroethers

The experiment described in Example 10 was repeated with the followingperfluoroethers: perfluoromethylbutyl ether (PFMBE),perfluoro-4-methyl-tetrahydrofuran (PMTH) and perfluorotetrahydropyran(PFTH). Transfection data is shown in Table 9 below. Table 9 shows thatperfluoroethers perform similar to perfluorocarbons of the previousexamples. Increased levels of perfluoroethers do not appear to have adetrimental effect on transfection and expression. Each of theperfluoroethers enhance transfection greater than 100% over the controllipid (DODO). TABLE 9 Lipids Perfluoroether PFE (vol) CAT expressionStd. Dev. (none) none — −54.773 4.957 DODO none — 1490.801 183.278 DODOPFMBE 0.125 mls 3578.363 55.702 DODO PFMBE 0.250 mls 3167.014 246.912DODO PFMBE 0.500 mls 3509.693 109.497 DODO PFMTH 0.250 mls 3424.94525.837 DODO PFMTH 0.500 mls 3373.024 36.736 DODO PFTH 0.250 mls 3306.02967.332 DODO PFTH 0.500 mls 3344.886 235.041

In all cases above the cell line is HeLa, and the lipid coating for theperfluoroether dioleyl-glycero-3 phosphocholine. CAT expression unitsare ng/ml protein. Std. dev.—standard deviation, PFE(vol)—perfluoroether in volume.

Example 16 Transfection Efficiency with Perfluorohexane

The experiment of Example 10 was repeated in HeLa cells using DMRIE-C,DODO, DPDO, or other commercial cationic lipids with and withoutperfluorohexane. The results are shown in Table 10 below. Table 10 showsthat perfluorocarbons are effective with a variety of lipids. Indeed,the enhancement of expression is independent of the type of lipid used.DODO+PFC6 results in about 8 to about 10 fold increase in expression,DMRIE-C results in about a 40% enhancement of expression, and DPDOresults in about a 4 fold increase. TABLE 10 CAT Lipids PFC PFC (vol)expression Std. Dev. (none) none — −5.608 5.015 DMRIE-C none — 1615.09679.088 DMRIE-C PFC6 0.125 mls 2118.489 72.325 DODO none — 611.594228.548 DODO PFC6 0.250 mls 6331.664 443.727 DODO PFC6 0.500 mls4829.148 379.244 DPDO none — 426.652 238.019 DPDO PFC6 0.125 mls1675.285 1146.279 Lipofectamine none — −8.891 1.895 Lipofectin none —100.542 56.863

DODO—dioleyl-glycero-3 phosphoethylcholine. CAT expression units areng/ml protein. DPDO—dipalmitoyl-glycero-3 phosphocholine, Std.Dev.—standard deviation, PFC (vol)—perfluorocarbon in volume.

Example 17 Effect of Ultrasound Alone on DMRIE and DODO VesicleTransfection

The experiments described above in the previous examples were repeatedusing the DMRIE, DODO, and no lipids, in each case without an organichalide, using the same procedures set forth above in Example 16. TABLE11 US Expression Lipids OH (sec.) Actual SD none none 0 76 4 DMRIE none0 426 49 DMRIE none 5 1450 317 DMRIE none 30 1204 120 DODO none 0 2719102 DODO none 5 4073 61 DODO none 30 3914 53

In all cases above the cell line is HeLa. Units of DNA expression areng/ml protein. Ultrasound was applied at 0.5 W/cm², 10% duty cycle.OH—organic halide, PFC—perfluorocarbon, US (sec.)—ultrasound in seconds,SD—standard deviation.

Example 18 Effect of Poly-L-Lysine (in Place of Lipids) on Transfectionwith Perfluorohexane

The experiments described above in the previous examples were repeatedusing the DODO with or without a fluorinated organic halide, Poly-L-Lyswith or without a fluorinated organic halide, and no lipids and noorganic halide, using the same procedures set forth above in Example 16.TABLE 12 CAT Carrier OH OH (vol) expression Std. Dev. (none) none —1.378 1.012 DODO none — 2190.158 684.125 DODO PFC6 0.125 3322.927 39.520Poly-L-Lys none — 37.901 2.072 Poly-L-Lys PFC6 0.125 50.909 5.128

DODO—dioleyl-glycero-3 phosphoethylcholine. CAT expression units areng/ml protein. OH—organic halide, OH (vol)—organic halide in volume inmls, Std. Dev.—standard deviation.

Example 19 Comparison of Different Organic Halides as TransfectionEnhancers

The experiment was carried out as in Example 10 using HeLa cells,dioleyl-glycero-3-phosphoethanolamine (DOPE) as the lipid carrier andeither perfluoropentane (PFC₅), 1-bromoperfluorooctane(perfluoro-octylbromide, BrPFC₈), perfluorodecane (PFC₁₀) orperfluorohexane (PFC₆). The Table 13 illustrates the transfectionresults. TABLE 13 OH vol CAT Lipids OH (in mls) expression Std. Dev.(none) none — −11.434 1.478 DOPE none — 295.447 18.828 DOPE PFC5 0.1251204.762 198.020 DOPE PFC5 0.250 900.344 247.634 DOPE PFC5 0.5001089.989 245.888 DOPE BrPFC8 0.125 276.236 57.468 DOPE BrPFC8 0.250248.652 26.723 DOPE BrPFC8 0.500 114.175 65.473 DOPE PFC10 0.125 839.263302.202 DOPE PFC10 0.250 811.678 171.344 DOPE PFC10 0.500 1186.044322.248 DOPE PFC6 0.125 1108.215 164.989 DOPE PFC6 0.250 829.904 156.464DOPE PFC6 0.500 441.745 33.219OH—organic halide,OH (vol)—organic halide in volume,Std. Dev.—standard deviation.

The above results indicate that liquid (perfluorohexane orperfluorodecane)perfluorocarbons work as well as perfluorocarbons whichwould undergo at least a partial liquid to gas phase transition atphysiological temperatures (perfluoropentane). Only the brominatedcompound, BrPFC₈, was markedly less effective in enhancing transduction.

The data also indicate that a preferred quantity of perfluorocarbon fortransfection generally is in the range of 0.125 mls to 0.250 mls or fromabout 1% to 3% v/v.

Example 20 Transfection in the Presence of Fluorinated Surfactants

The experiment described in Example 10 is repeated except the lipids aresuspended in varying amounts (1.25% to 5%) of Zonyl® surfactant, (DuPont Chemical Co., Wilmington, Del.). In some samples perfluorohexane(0.125 mls, 0.250 mls or 0.500 mls) is shaken with the surfactant. Thesizes range from about 300 nm to about 900 nm. Transfection efficiencywhere samples are prepared from Zonyl® is about 10% to about 25% of thetransfection where samples are prepared without Zonyl®.

Example 21 Transfection with Cationic Lipid Carrier and Lipid Suspensionwith Organic Halides and with and without Ultrasound

The experiment described in Example 14 is modified such that the controllipid mixture is DPPC/DPPA/PEG-5000+perfluoropropane and results arecompared to transfection using a suspension of the cationic lipidDOTMA+perfluoropropane. After incubation ultrasound is applied as inExample 14 to some of the samples. Results similar to those obtained inExample 14 are observed.

Example 22 Transfection with Cationic Lipid Carrier and Lipid Suspensionwith Ultrasound and 1-Bromononafluorobutane

Cationic liposomes are prepared from dioleyolyethylphosphocholine andDOPE at a concentration of 1 mg/ml lipid. To this is added 0.125 μl1-bromo-nonafluorobutane (BNFB). The mixture is agitated with aWig-L-Bug for 60 seconds and the resulting vesicles are extruded with a0.8 micron filter. The resulting BFNB filled cationic liposomes are thencomplexed with ribozymal RNA (hammerhead motif) encoding catalytic RNAspecific for vascular endothelial growth factor (VEGF) at a lipid to RNAratio of 1:6 using conventional methods. The liposomal RNA preparation(1.0 ml) is injected IV into a patient with diabetic retinopathy. A 5megahertz ultrasound transducer is placed on the patient's anesthetizedcornea using a silicone acoustic coupling gel. Ultrasound energy isfocused onto the patient's retina using 2.0 Watts and a 10% duty cycle.As the liposomes enter the region of ultrasound the gaseous precursorexpands and pulsates. Local shock waves are created. The ribozyme RNA isdelivered to the endothelial cells in the region of ultrasoundapplication the therapeutic RNA construct enters the target endothelialcells. Catalytic RNA then causes reduction in VEGF production. Thepatient's retinal deterioration is halted and blindness is avoided.

Example 23 In Vivo Transfection Using Perfluorocarbons

Liposomes were prepared from dipalmitoylethylphosphocholine (DPEPC) anddioleoylphosphatidylethanolamine (DOPE). Both lipids were purchased fromAvanti Polar Lipids (Alabaster, Ala.). Lipofectin® and Lipofectamine®and DMRIE-C® were obtained in a ready to use form from Life TechnologiesInc. (Gaithersburg, Md.). Plasmid pCMVCAT which contains the humancytomegalovirus promoter and the chloramphenicol acetyl transferase genewas provided by Life Technologies (Gaithersburg, Md.). Plasmid DNA wasprepared by large scale extraction from E. coli and purified by CsClbanding by Lofstrand Labs Limited (Gaithersburg, Md.). In a preliminaryset of experiments the ratios of the two lipids as well as the ratio oflipid to DNA was optimized by measuring the levels of gene expression inHeLa cells following transfection with the pCMVCAT plasmid usingDPEPC/DOPE in a ratio of 1:1, with a lipid to DNA ratio of 6:1 (thelipid and DNA combination collectively being referred to as DPDO). Theliposomes were prepared by suspending the dried lipids in water andlyophilizing the mixed lipids and resuspending the lipids in water. Therehydrated lipids were agitated at about 2,000 r.p.m. on a dentalamalgamator (Heavy duty #6 Wig-L-Bug Crescent Dental, Lyons, Ill.) for 5minutes and then extruded through two 0.8 micron polycarbonate filters(Nucleopore Costar, Cambridge, Mass.) using an Extruder Device (LipexBiomembranes, Vancouver, B.C., Canada) at about 30 psi. For preparationof perfluorocarbon (PFC) filled liposomes, 100 microliters, 50, 25 or12.5 microliters of PFC was added to the lipid solution prior to shakingon the dental amalgamator. PFC's which were tested includedperfluoropentane, perfluorohexane, 1-bromononafluorobutane,perfluorooctylbromide and perfluoromethylbutylether.

In vivo experiments were carried out in 50 male Balb/C mice. The bodyweight of the mice was 15-20 g. The lipoplex was formed in the samemanner as for cell transfection using the DPEPC/DOPE lipid mixture. Theperfluorocarbon used in this experiment was perfluorohexane. The mixturewas injected intramuscularly at a does of 200%1 per hind leg. Theultrasound (US) was applied at 1 W/cm² for one minute to each leg in theanimals that received ultrasound. The animals were held for 2 days andthen euthanized by carbon dioxide asphyxiation. The hind legs wereremoved from the animals and the muscle collected. The muscle was frozenin liquid nitrogen and ground in a mortar and pestle. The tissue wastransferred to a pre-weighed 50 ml conical tube and the tissue weightwas recorded. The tissue was then placed into one ml of the lysis bufferfrom the CAT ELISA kit. The CAT ELISA was then carried out according tothe manufacturer's protocol. The data was transferred into MicrosoftExcel and converted into ng CAT protein per gram of tissue. Statisticalanalysis was carried out using the JMP 3.1.5 statistical analysispackage for the Macintosh.

The results are shown in FIG. 7. As the figure shows, the use of aperfluorocarbon (with or without ultrasound) greatly enhanced CATexpression in mice.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

1. A method for delivering a compound into a cell comprisingadministering to the cell a composition which comprises the compound tobe delivered and an organic halide. 2-104. (canceled)